11.6: Vesicular Transport - Biology

In addition to protein processing, the ER and Golgi also take care of some types of protein transport. Vesicles (membrane-bound bubbles, essentially) pinch off from the ER, Golgi, and other membranous organelles, carrying with them whatever soluble molecules were inside the fluid that was enclosed as well as any molecules embedded in that section of membrane. These vesicles then catch a ride on a molecular motor such as kinesin or myosin, and travel along the cytoskeleton until they dock at the appropriate destination and fuse with the target membrane or organelle. In general, vesicles move from the ER to the cis Golgi, from the cis to the medial Golgi, from the medial to the trans Golgi, and from the trans Golgi to the plasma membrane or other compartments. Although most movement is in this direction, there are also vesicles that move back from the Golgi to the ER, carrying proteins that were supposed to stay in the ER (e.g. PDI) and were accidentally scooped up within a vesicle.

The formation of vesicles is dependent on coat proteins that will, under proper conditions, self-assemble into spherical cages. When associated with transmembrane proteins, they can pull the attached membrane along into a spherical shape also. The major types of coat proteins used in vesicle formation are COPII, COPI, and clathrin.

COPII coat proteins form the vesicles that move from ER to Golgi. COPI coat proteins are used between parts of the Golgi apparatus as well as to form vesicles going from the Golgi back to the ER. Finally, clathrin is used to form vesicles leaving the Golgi for the plasma membrane as well as for vesicles formed from the plasma membrane for endocytosis.

Clathrin (Figure (PageIndex{17})) is the best described of the three, and the vesicular coats are made from arrangements of clathrin triskelions (from Greek, meaning three-legged). Each triskelion is composed of three heavy chains joined together at the C-terminus, and three light chains, one associated with each heavy chain. The heavy chains of different triskelions interact along the length of their heavy chain “legs” to create a very sturdy construct. The light chains are unnecessary for vesicle formation, and are thought to help prevent accidental interactions of clathrin molecules in the cytoplasm.

There is significant similarity between the vesicle formation mechanisms using these different coat proteins, beginning with the recruitment of ARF1 (ARF stands for ADP ribosylation factor, which has nothing to do with its function here) to the membrane. This requires the ARNO-facilitated exchange of a GTP for GDP (ARNO is ARF nucleotide binding site opener). Once ARF1 has bound GTP, the conformational change reveals an N-terminal myristoyl group which inserts into the membrane. Both COPI and clathrin-coated vesicles use ARF1 and ARNO, but COPII uses similar proteins called Sar1p and Sec12p.

Figure (PageIndex{18}). COP-coated vesicles

The ARF1 (or Sar1p) is used to recruit adapter proteins that bind to the “tail” end of membrane-bound receptor proteins. The business end of these receptors binds to car- go molecules that need to be packaged into the vesicle. The adapter proteins act as the link between the membrane (through the receptors) and the coat proteins. For clathrin, the adapter proteins are AP1 for trans-Golgi-derived vesicles and AP2 for endocytic vesicles. For COPI vesicles, the approximate homologues are the β-, γ-, δ-, and ζ- COPs while the COPII system uses Sec23p and Sec24p.

Finally, the adapters link to the actual coat proteins: clathrin, α- or ε- COP, Sec13p and Sec31p. What these proteins all have in common is that spontaneously (i.e. without any requirement for energy expenditure), they self-assemble into cage-like spherical structures. Under the electron microscope, the clathrin-coated vesicles are more sharply defined and the hexagonal and pentagonal shapes bounded by the clathrin subunits give the vesicle a “soccer ball” look. COP coatamer-coated vesicles are much fuzzier in appearance under EM.

All three types of vesicle coat proteins have the ability to spontaneously associate into a spherical construct, but only the COPI and COPII coated vesicle also spontaneously “pinch off” the membrane to release the vesicle from its originating membrane. Clathrin-coated vesicles require an external mechanism to release the vesicle (Figure (PageIndex{19})).

Once the vesicle has almost completed, there is still a small stalk or neck of membrane that connects the vesicle to the membrane. Around this stalk, dynamic GTP molecules aggregate in a ring/spiral construction. Dynamin molecules are globular GTPases that contract upon hydrolysis of GTP. When they associate around the vesicle stalk, each dynamin protein contracts, with the combined effect of constricting the stalk enough that the membrane pinches together, sealing off and releasing the vesicle from the originating membrane.

Although lipids and membranes were discussed in chapter 4, we neglected to discuss the location of their syntheses in eukaryotes. As Figure (PageIndex{20}) indicates, the synthesis of certain types of lipids is segregated and exclusive. Glycerophospholipids are primarily formed in the endoplasmic reticulum, although they are also made in mitochondria and peroxisomes. In contrast, sphingolipids are not made in the ER (though their ceramide precursors are) in mammals, the necessary enzymes are found in the lumen of the cis and medial Golgi. There is evidence of anterograde and retrograde vesicular traffic between the various Golgi and ER compartments, which would theoretically indicate a redistribution of lipid types. However, the sphingolipids tend to aggregate into lipid rafts and seem to be more concentrated in anterograde-moving vesicles.

The coat proteins come off shortly after vesicular release. For clathrin, the process involves Hsc70, an ATPase. However, for COPI or COPII coated vesicles, hydrolysis of the GTP on ARF/Sar1p appears to weaken the coat protein affinity for the adapters and initiates uncoating. The GTPase activator is ARF GAP (or Sec23p) and is an integral part of the COP I (or II) coat.

The vesicles carry two categories of cargo: soluble proteins and transmembrane proteins. Of the soluble proteins, some are taken up in the vesicle by virtue of being bound to a receptor. Other proteins just happen to be in the vicinity and are scooped up as the vesicle forms. Occasionally, a protein is taken up that was not supposed to be; for example, PDI may be enclosed in a vesicle forming from the ER. It has little function in the Golgi, and is needed in the ER, so what happens to it? Fortunately, PDI and many other ER proteins have a C-terminal signal sequence, KDEL (Lysine-Aspartic Acid-Glutamic Acid-Leucine), that screams “I belong in the ER.” This sequence is recognized by KDEL receptors inside the Golgi, and binding of the KDEL proteins to the receptors triggers vesicle formation to send them back to the ER.

Secretory vesicles have a special problem with soluble cargo. If the vesicle was to rely simply on enclosing proteins within it during the formation process, it would be difficult to get high concentrations of those proteins. Many secreted proteins are needed by the organism quickly and in significant amounts, so there is a mechanism in the trans Golgi for aggregating secretory proteins. The mechanism uses aggregating proteins such as secretogranin II and chromogranin B that bring together the target proteins in large concentrated granules. These granins work best in the trans Golgi milieu of low pH and high Ca2+, so when the vesicle releases its contents outside of the cell, the higher pH and lower Ca2+ breaks apart the aggregates to release the individual proteins.

There is a consistent pH change during the maturation of the Golgi, so that as we go from ER to Golgi, each compartment has a progressively lower (more acidic) lumenal pH.

Finally, there is the question of targeting the vesicles. The vesicles are much less useful if they are tossed on a molecular freight train and dropped off at random. Therefore, there is a docking mechanism that requires a matching of the v-SNARE protein on the vesicle’s cytoplasmic surface and the t-SNARE on the cytoplasmic surface of the target membrane. Fusion of the vesicle to the membrane only proceeds if there is a match. Otherwise, the vesicle cannot fuse, and will attach to another molecular motor to head to another, hopefully correct, destination. This process is aided by tethering proteins which initially make contact with an incoming vesicle and draw it close enough to the target to test for SNARE protein interaction. Other proteins on the vesicle and target membranes then interact and if the SNAREs match, can help to “winch” the vesicle into the target membrane, whereupon the membranes fuse. An important rule of thumb to understanding vesicular fusion and also the directionality of membrane proteins and lipids, is that the cytoplasmic-facing side of a membrane is always going to be facing the cytoplasm. Therefore a protein that is eventually found on the outer surface of the cell membrane will have been inserted into the lumenal surface of the ER membrane to begin with.

More specifically, as a vesicle approaches the target membrane, the tethering protein Rab-GTP, which is linked to the target membrane via a double geranylgeranyl lipid tail, loosely associates with the vesicle and holds it in the vicinity of the target membrane to give the SNARES a chance to work. The v-SNAREs and t-SNAREs now have the opportunity to interact and test for a match. Recently, the SNAREs have been renamed R-SNAREs and Q-SNAREs, respectively, based on conserved arginine and glutamine residues. In addition to these two primary SNAREs, at least one other SNARE is involved, together forming a bundle of four α-helices (four, not three, because at least in the best studied example, one of the SNAREs is bent around so that two of its alpha-helical domains participate in the interaction. The four helices wrap around each other and it is thought that as they do so, they pull the vesicle and the target membrane together.

The tetanus toxin, tetanospasmin, which is released by Clostridium tetani bacteria, causes spasms by acting on nerve cells, and preventing neurotransmitter release. The mechanism for this is that it cleaves synaptobrevin, a SNARE protein, so that the synaptic vesicles cannot fuse with the cell membrane. Botulinum toxin, from Clostridium botulinum, also acts on SNAREs to prevent vesicle fusion and neurotransmitter release, although it targets different neurons and so has the opposite effect: tetanus is caused by preventing the release of inhibitory neurotransmitters, while botulism is caused by preventing release of excitatory neurotransmitters.

Vesicle transport v-SNARE 11

<p>The annotation score provides a heuristic measure of the annotation content of a UniProtKB entry or proteome. This score <strong>cannot</strong> be used as a measure of the accuracy of the annotation as we cannot define the 'correct annotation' for any given protein.<p><a href='/help/annotation_score' target='_top'>More. </a></p> - Experimental evidence at protein level i <p>This indicates the type of evidence that supports the existence of the protein. Note that the 'protein existence' evidence does not give information on the accuracy or correctness of the sequence(s) displayed.<p><a href='/help/protein_existence' target='_top'>More. </a></p>

Select a section on the left to see content.

Vesicular transport mediates the uptake of cytoplasmic proteins into mitochondria in Drosophila melanogaster

Mitochondrial aging, which results in mitochondrial dysfunction, is strongly linked to many age-related diseases. Aging is associated with mitochondrial enlargement and transport of cytosolic proteins into mitochondria. The underlying homeostatic mechanisms that regulate mitochondrial morphology and function, and their breakdown during aging, remain unclear. Here, we identify a mitochondrial protein trafficking pathway in Drosophila melanogaster involving the mitochondria-associated protein Dosmit. Dosmit induces mitochondrial enlargement and the formation of double-membraned vesicles containing cytosolic protein within mitochondria. The rate of vesicle formation increases with age. Vesicles originate from the outer mitochondrial membrane as observed by tracking Tom20 localization, and the process is mediated by the mitochondria-associated Rab32 protein. Dosmit expression level is closely linked to the rate of ubiquitinated protein aggregation, which are themselves associated with age-related diseases. The mitochondrial protein trafficking route mediated by Dosmit offers a promising target for future age-related mitochondrial disease therapies.

Conflict of interest statement

The authors declare no competing interests.


Fig. 1. Mitochondrial size and rate of…

Fig. 1. Mitochondrial size and rate of double-membraned intramitochondrial vesicle formation increases with age.

Fig. 2. Knocking down a mitochondria-associated protein,…

Fig. 2. Knocking down a mitochondria-associated protein, Dosmit, results in significant differences in mitochondrial size…

Fig. 3. Dosmit protein expression levels increase…

Fig. 3. Dosmit protein expression levels increase in wild-type flies throughout natural aging.

Fig. 4. Dosmit-induced mitochondrial enlargement and formation…

Fig. 4. Dosmit-induced mitochondrial enlargement and formation of intramitochondrial vesicles in larvae and adult flies.

Fig. 5. Ectopic expression of Dosmit induces…

Fig. 5. Ectopic expression of Dosmit induces the formation of intramitochondrial vesicles containing cytosolic proteins.

15% of the cytosolic GFP was internalized in mitochondria when Dosmit (Dsm) was ectopically expressed. N = 2. e Localization of gold-labeled GFP within the intramitochondrial vesicles of flight muscles from flies ectopically expressing Dosmit (Mef2 > Dosmit + GFP, arrowheads) and from mitochondria of flight muscles expressing GFP without ectopic expression of Dosmit (Mef2 > GFP) and control mitochondria (Mef2/+). Color panels are confocal microscopy images gray-scale panels are TEM images. Scale bars: 0.5 μm. f GFP–gold particles localized inside and outside the intramitochondrial vesicles in muscles from flies ectopically expressing Dosmit (Mef2 > Dosmit + GFP). N = 7 and 7 from left to right bars (mean ± SD). Statistical test: Two-tailed Mann–Whitney U-test. (***p < 0.001). g Three-dimensional imaging of Mef2 > Dosmit + GFP fly mitochondria, with cytosolic GFP internalizing in the mitochondria. Source data are provided as a Source Data file.

Fig. 6. Ectopic expression of Dosmit induces…

Fig. 6. Ectopic expression of Dosmit induces the formation of intramitochondrial vesicles containing Tom20.

Fig. 7. Aged mitochondria exhibit internalization of…

Fig. 7. Aged mitochondria exhibit internalization of cytosolic content, but no vesicles appear in aging…

Fig. 8. The mitochondria-associated protein, Rab32, colocolized…

Fig. 8. The mitochondria-associated protein, Rab32, colocolized with Tom20-HA and undergoes a protein–protein interaction with…

Fig. 9. Rab32 and Dosmit localization on…

Fig. 9. Rab32 and Dosmit localization on mitochondria is mutually dependent.

Fig. 10. The mitochondria-associated protein Rab32 mediates…

Fig. 10. The mitochondria-associated protein Rab32 mediates the Dosmit-induced enlargement of mitochondria.

Fig. 10. The mitochondria-associated protein Rab32 mediates…

Fig. 10. The mitochondria-associated protein Rab32 mediates the Dosmit-induced enlargement of mitochondria.

11.6: Vesicular Transport - Biology

C2006/F2402 '11 -- Outline For Lecture #6

(c) 2011 Dr. Deborah Mowshowitz , Columbia University, New York, NY . Last update 02/06/2011 02:55 PM

Handouts: 6A-- Transport of glucose through body (gif) 6A-- pdf
6B -- RME (gif) 6B --RME (pdf)
6C -- Structure of Capillaries & Transcytosis
(Posted on Courseworks).
Here are links for a diagram of a capillary, a diagram of transcytosis, and an electron micrograph of a capillary.

I. Putting all the Methods of Transport of Small Molecules Together or What Good is All This?

A. How glucose gets from lumen of intestine → muscle and adipose cells. An example of how the various types of transport are used. (Handout 6A) Steps in the process:

1. How glucose exits lumen. Glucose crosses apical surface of epithelial cells primarily by Na + /Glucose co-transport. (2 o act. transport).

2. Role of Na+/K+ pump. Pump in basolateral (BL) surface keeps Na + in cell low, so Na + gradient favors entry of Na + . (1 o act. transport)

3. How glucose exits epithelial cells.

a. Glucose (except that used for metabolism of epithelial cell) exits BL surface of cell by facilitated diffusion = carrier mediated transport.

b. Transporter protein = GLUT2 (more details on GLUT family of proteins below).

c. When glucose leaves cells it enters the interstitial fluid = IF = fluid in between body cells.

4. How glucose enters and leaves capillaries -- by simple diffusion through spaces between the cells. Cells surrounding capillaries in most of body are notjoined by tight junctions.

a. Material does NOT enter capillaries by diffusion across a membrane. Material diffuses through liquid in spaces (pores) between the cells.

b. For structure of capillaries, see handout 6C, bottom. (Also see links at start of lecture.) Pictures are provided on handout since function is hard to understand without the anatomy. Picture shows how endothelial cells surround capillary lumen, forming pores between cells. Pores allow diffusion (of glucose and other small hydrophilic molecules, but not proteins) in and out of capillary.

c. Blood brain barrier: Capillary cells in brain are joined by tight junctions -- there are no pores (spaces between cells) so material cannot diffuse in and out of capillaries in brain. Click here for details on the BBB. (fyi only).

5. How glucose enters body cells

a. Glucose enters cells by facilitated diffusion = carrier mediated transport using a GLUT protein.

b. Carrier is permanently in cell membrane in many cell types (brain, liver). See below on GLUT transporters.

c. Carrier (GLUT 4) is only "mobilized" that is, inserted into membrane (by fusion of vesicles as explained previously) in some cell types (adipose & muscle) in presence of insulin.

6. Role of glucose phosphorylation. Conversion of G → G-6-phosphate traps G inside cells.

For additional examples of the uses of the various types of transport processes, see Becker fig. 8-1 & 8-2. For pictures of steps 1-3, see or

Note both of these come from classes with extensive on line notes. The biochem course includes several animations of transport proteins.

B. How Glucose Reaches Body Cells -- Another look at handout 6-A. The steps in the process are described above in the order in which they occur. Here is a summary with the focus on the various types of transport involved.

1. Role of Active transpo rt -- Needed to get glucose from lumen to inside of epithelial cell.

a. Primary active transport -- Na + /K + pump keeps intracellular [Na + ] low.

b. Secondary active transport -- Glucose enters epithelial cells by Na + /Glucose co-transport

2. Role of Passive Transport & Phosphorylation (of glucose)

a. Passive Transport -- Used to move glucose the rest of the way -- out of epithelial cells, in & out of capillaries, and into body cells.

b. Phosphorylation of glucose -- Used in the body cells to keep the free glucose level at the "end of the road" low, and ensure that the glucose gradient is "downhill" from epithelial cells to capillaries to body cells.

3. Role of Diffusion: Glucose and other small molecules (but not macromolecules) diffuse in and out of capillaries through the liquid filled spaces between the cells, not by diffusing across the cell membrane.

Most proteins are too big to enter or leave capillaries by diffusion. Most proteins enter and leave by transcytosis shown on handout 6C and explained below.

4. Role of Channels: None are shown on the handout, but glucose can pass from one epithelial cell to another through gap junctions.

5 . Role of GLUT transporters (another protein/gene family)

a. GLUT proteins are responsible for passive transport of glucose. All passive glucose transport across membranes (that is carrier mediated) depends on a family of proteins called GLUT 1, GLUT 2, etc. (GLUT = Glucose transporters)

b. Different family members (genes and proteins) are expressed in different cell types. GLUT 1 protein is found in plasma membrane of RBC & most other cells, GLUT 2 protein on BL surface of intestinal epithelial cells, GLUT 4 protein in muscle and adipose tissue, etc. (Note all genes for all proteins are present in all these cell types -- DNA is the same!)

c. All the genes and corresponding proteins are similar, but have significant structural and functional differences. This is another example of a gene/protein family. All the proteins have a similar overall structure -- 12 transmembrane segments, COOH and amino ends on intracellular side of membrane, etc. For a picture click here. For a diagram and table go to

d. Position & Action of GLUT 4 is insulin dependent. GLUT 4 is the only insulin dependent member of the family. Insulin triggers insertion of GLUT 4 protein into the plasma membrane, by triggering vesicle fusion, as explained previously. All the other proteins are located constitutively in their respective membranes.

e. Direction of transport. Note that one member of this family (GLUT 2) is responsible for ferrying glucose OUT of epithelial cells different members are responsible for helping glucose ENTER most other cells. All family members bind glucose on one side of the membrane, change conformation and release glucose on the other side of the membrane. Which way the glucose goes (in or out) depends on the relative concentrations of glucose on the two sides of the respective membrane, not on which GLUT protein is used. (See problem 2-12 C.)

f. SGLT proteins are responsible for active transport of glucose. (SGLT = Sodium -glucose transporters). SGLT proteins make up a different protein family. The members of this family are responsible for active transport of glucose across membranes. (See problem 2R-2.)

g. All transport of glucose into and out of cells requires a transport protein. Protein can be a carrier, pump, or channel. Transport into capillaries by diffusion between the cells does not require a protein transporter.

Try problem 2-9 & 2-12.

II. Ways that Big Molecules Enter Cells -- Types of Endocytosis. In all cases net effect is that cell membrane folds in and pinches off, forming a vesicle in the cytoplasm that contains material from the outside.

A. Pinocytosis = bulk phase endocytosis no receptor. Cells take in random samples of surrounding fluid containing a random selection of extracellular substances.

B. Phagocytosis -- in specialized cells only -- extensions of cells (pseudopods) reach out and engulf solids. See Becker fig. 12-14. Vesicle that is formed is called a phagocytic vesicle (or vacuole) or phagosome. Requires MF.

C. RME = receptor mediated endocytosis. Cells take in specific substances from surrounding fluid using a receptor. See Becker fig. 12-15 (diagram) & 12-16 (micrograph). Different cell types have different combinations of receptors.

III. RME -- Receptor Mediated Endocytosis

A. General and/or important Features.

1. Receptors -- Need specific receptor for each substance (or class of closely related substances) to be transporte d.

2. Concentrates substances transported -- usually moves them up their gradient.

3. Requires energy -- multiple stages in process use ATP or GTP. Energy must be required because substances move against their gradients. Energy is required to form the vesicles and to process and/or transport the vesicles inside the cell.

4. Role of clathrin -- A peripheral membrane protein is needed to deform membrane and allow vesicles to form -- provides a coat. (See Becker figs. 12-15 to 2-18 and/or Sadava fig. 6.19 (5.17) Other proteins are required as well, but will not be discussed.

a. Clathrin is coat protein for vesicles forming from plasma membrane and trans-Golgi*.

b. Budding of other membranes involve different "coat" proteins. Best known are COPI & COPII which are involved in ER-Golgi transport. (See Becker for details if interested. Types of coats are summarized in table 12-2.)

*Note on terminology: Trans side of Golgi = "far end" = side away from nucleus and ER = last part that proteins travel through as they are processed in Golgi. Also called "TGN" for "trans Golgi Network." See Sadava fig. 5.11 (4.11) or Becker fig. 12-8 for labeled pictures. More details on structure and function of Golgi later.

5. It's a cycle -- Exocytosis balances endocytosis so cell surface area stays the same. See Sadava fig. 6.18 (5.16) or Becker fig. 12-15. For LDL receptor, it takes about 10-20 minutes for one "round trip."

6. Topology -- material can enter and/or exit cell without being in contact with cytoplasm. Material can remain inside a vesicle or outside cell at all times. (See Transcytosis.)

7. Possible fates of endocytosed material -- Where does vesicle go? Where do receptor &/or ligand end up?

a. Degradation -- vesicle fuses with lysosomes and contents are degraded.

b. Return to surface -- material recycles -- vesicle fuses with plasma membrane.

c. Sorting -- not everything in the vesicle may go to the same place. Vesicle may fuse with endosome (sorting vesicle), and different parts of the endocytosed material may be directed to different destinations. (More details on a-c below.)

d . Transcytosis -- vesicle crosses cell and fuses with opposite cell surface. For examples see handout 6C or diagram of transcytosis (shows how antibodies enter lumen)

(1). Requires Receptor: Transcytosis requires a receptor for each substance transported. Receptor is not shown on 6C but is clearly shown on diagram of transcytosis

(2). Transcytosis = end ocytosis + exocytosis = 2 steps

(a). Material binds to receptor and is endocytosed on one surface of the cell.

(b). Vesicle moves across cell and material is exocytosed on a different surface.

(3). Function. Can be used to move proteins, across a cell, in either direction. See examples above or RP3.

B. Stages of Cycle (Numbers match steps on handout 6B .) Click here for animation.

  • A single endosome may contain many different receptors and ligands, and different ones are sorted differently. (Some examples are given in detail below.)
  • The uncoated, acidified vesicle can be called an endosome, early endosome, or a sorting vesicle.
  • Acidification requires energy to run proton pump -- to move H + into vesicle at expense of ATP. Pump is in membrane of vesicle.

Note: Details of sorting and recycling -- the remaining steps -- vary with material endocytosed. More details below for individual cases.

(7). Endosome splits . The substance we are following, and/or its receptor, can end up in either half.

In example shown on handout, one half gets the receptor and one half gets the ligand, as is the case for LDL. Other examples will be discussed in class and are outlined in detail below.

Note: endosome may not simply split in one step process of sorting may be gradual. Pieces of different composition may gradually bud off as internal composition of remainder changes.

(8) . What Happens to the Different Parts of the Endosome?

8A. Fate of vesicle with materials to be recycled (receptors and/or carriers) -- this vesicle fuses with plasma membrane in step 9. (In case of LDL, this vesicle would contain the receptor for LDL.)

8B. Fate of vesicle with material that remains inside the cell -- Vesicle delivers contents to appropriate cell compartment. (For LDL, vesicle delivers LDL to lysosomes, so material is degraded.)

(9). Exocytosis occurs -- returns receptors and/or other components to the plasma membrane or outside of cell.

Try Problem 2-6.

C. Some Specific Examples

1. LDL (Low density lipoprotein ) -- receptor recycled, but ligand (including protein part) degraded. See Becker, Box 12B or text of Sadava Ch. 51.4 (50.4). Many of LDL details may have been included in general case, but are summarized below. Click here for a picture of LDL.

a. What is LDL? A lipoprotein particle containing cholesterol esters + some other lipids + a protein. Particle contains esterified cholesterol covered by monolayer of amphipathic lipid (phospholipid plus some unesterified cholesterol) + one molecule of protein (apoprotein B or apoB).

b. Why LDL?

(1) Why a monolayer on outside? Solubility. Cholesterol is insoluble in blood. (Too hydrophobic.) Need a way to ferry cholesterol through blood and into cell -- Cholesterol transport requires formation of particle with hydrophilic surface

(2) Why a protein (apoB)? For binding to cell surface receptor (LDL receptor). A protein is needed as ligand to bind to receptor.

(3) Summary of Roles of Parts of LDL:

(1). Protein (apoB) = Ligand = what actually binds LDL receptor = protein part of LDL

(2). Cholesterol -- What cell actually needs is the cholesterol part (for building its membranes &/or hormone synthesis).

c. Receptor, but not protein part of LDL, is recycled. Note: there are 2 separate proteins here that are easily confused

(1) Receptor protein on the cell surface = LDL receptor = binds LDL and allows uptake of cholesterol

(2) Protein in LDL (apoB) = ligand for LDL receptor = part of LDL and helps carry cholesterol through the blood.

d. Receptor and apoB are separated inside sorting vesicles/endosomes. All of LDL (including protein) stays together separates from receptor

e. Need lysosomes to degrade LDL protein and release cholesterol (cholesterol esters in LDL must be split for cholesterol to be used). How does LDL reach lysosomes? Through fusion of vesicles. Either:

(1). Vesicles/endosomes holding substrate fuse with pre-existing lysosomes, or

(2). V esicles with substrate fuse with vesicles from Golgi carrying newly made hydrolases to form new lysosomes. (More details on how hydrolases pass through the Golgi and are targeted to lysosomes to be discussed later.)

f. Function of LDL uptake -- to supply a nutrient (cholesterol).

g . Current terminology: relationship of early endosomes, late endosomes & lysosomes. Note: Most of this is FYI. In this course, the term "endosomes" will be used for both early and late endosomes.

(1). Early endosome = sorting vesicle. Term is used differently by different authors. Can be "early" on pathway into cell (by endocytosis) and/or "early" on pathway from Golgi to lysosomes. Therefore, early endosomes can mean:

(a) Uncoated & acidified vesicles from invagination of plasma membrane carrying newly endocytosed material,

(b). Vesicles coming from Golgi carrying newly made proteins (more on this later).

(2). Late endosome = vesicle containing hydrolytic enzymes destined for lysosomes (but not yet activated) plus potential substrate. More acidic than early endosome. Material not destined for lysosomes has been jettisoned. Formed by maturation of early endosome.

(3). Lysosomes = vesicle containing active hydrolytic enzymes and substrate. More acidic than late endosome. Formed by maturation of late endosome and/or fusion with pre-existing lysosome.

(4). Older terminologyfound in some texts (FYI only):

(a). Primary lysosome = vesicle with hydrolytic enzymes only.

(b). Secondary lysosome = enzymes + substrate = result of fusion of primary lyso. + another vesicle containing substrate.

2. EGF (Epidermal Growth Factor) -- all the protein involved (ligand + receptor) is degraded

a. No separate protein ligand required EGF is a protein -- unlike cholesterol, or Fe (see case below). EGF itself binds to receptor = ligand for cell surface receptor & substance that will be transported into the cell.

b. Function of uptake -- to regulate signaling. EGF is a signaling molecule. Uptake turns off signal and down regulates receptors (reduces # of cell surface receptors).

c. Receptor not recycled -- Ligand (signal molecule) and receptor degraded together.

d. Need lysosomes (to degrade both receptor and ligand).

3. Fe/Transferrin -- none of the protein involved is degraded -- all recycled

a. What is transferrin? Fe needs a protein (like cholesterol needs apoB) for transport and binding to receptor protein (= ligand for cell receptor) is called transferrin .

b. Both apotransferrin & receptor are recycled .

c. No lysosomes needed -- iron is transported out of endosome (using transporter protein or channel in membrane) no protein is degraded .

d. Transferrin and receptor separate outside cell after recycled

(1). Fe/transferrin binds to receptor at neutral pH and enters cell by RME.

(2). Inside cell, Fe transported out of vesicle into cytoplasm, leaving apo-transferrin stuck to receptor ("apo" means without ligand, cofactor, etc.).

(3). Apo-transferrin (= transferrin without Fe) sticks to receptor at low pH (in endosome) but separates at neutral pH (outside cell). This is contrary to usual behavior -- Most ligands stick to receptors at neutral pH but separate at low pH found in endosome. (Low pH breaks many weak bonds.)

(4). Note that apo-transferrin and Fe/transferrin have different affinities for the receptor at neutral pH. Under these conditions (neutral pH), Fe/transferrin binds to the receptor, and apo-transferrin separates from the receptor.

e. Function of uptake -- to supply a nutrient (Fe).

D. For Reference: Compare & Contrast for the examples described above for transport of X

Transferrin LDL EGF
What's carried in (what is X)? Fe Cholesterol Growth Factor
Function of X Metabolism (Fe is cofactor for many proteins) Metabolism (cholesterol is a component of cell membranes used for hormone synthesis) Signal
Ligand (What binds to receptor?) Transferrin = apotranferrin + Fe LDL EGF
Does ligand include protein in addition to X? Yes (apotransferrin) Yes (ApoB) No
Ligand Fate (protein part) Recycled Digested Digested
Receptor Fate Recycled Recycled Digested
Do protein part of ligand & receptor separate inside cell? No Yes No
Lysosomes Involved? No Yes Yes
Where do ligand & receptor separate? Outside the cell In endosomes Not separated -- both degraded

IV . Labeling -- How do you follow material coming into the cell? See handout 6C, top.

A. Types of Labeling (using added tracers)

1. Continuous Labeling -- switch from regular, ordinary material to labeled material (material containing radioactivity, fluorescence, etc.) and follow what gets labeled first (with radioactivity, fluorescence, etc.), what gets labeled next, and so on. We will discuss radioactive labeling, but the principle is the same whether the label is radioactivity, fluorescence, etc.

2. Pulse-Chase Experiments -- supply radioactive material for a brief time (pulse) and then switch back to ordinary, non-radioactive material (chase). Follow where the radioactivity goes. The "pulse" passes through the cell like a mouse through a boa constrictor. Just as different parts of the boa constrictor bulge out temporarily as the mouse passes down the snake, so different parts of the cell become radioactive temporarily, one at a time, as the radioactive material passes through. Then as the "pulse" or the "mouse" passes on, each part will return to normal -- non radioactive or normal size, depending on whether we are referring to the cell or to the snake.

B. Detection -- How do you find where the radioactivity (or whatever tracer/label you used) is?

1. Autoradiography -- Cover a layer of labeled cells with photographic emulsion and count radioactive grains over each organelle or part of the cell. This method is similar to doing in situ assays, in that you examine intact cells to pin down the location of what you are looking for. See Becker, Appendix, A-17 (A-18) or Guide to Microscopy.

Note: Becker's Appendix (or Guide to Microscopy in 5th ed.) has a lot of useful background info on microscopic methods, including immunofluorescence, freeze fracture, etc.

For a picture of typical results, see fig. 12-10 of Becker (7th ed) or click here. These pictures were obtained by following newly made material out of the cell, not following it in. However the principle is the same -- it shows label in different cell parts at different times.

2. Fractionate First -- Break up labeled samples, fractionate into various organelles, and measure radioactivity in each fraction. This is similar to the "grind and find" procedure, in that you break up the cells, separate them into their parts, and test a solution or suspension of each part for what you are looking for.

To review labeling and RME, try problems 2-8 & 2-11 by now you should be able to do all the problems in problem set 2 & 2R.

Next Time: How do proteins get sorted to their proper place? How do molecules get in and out of the nucleus?

Brian Rymond

Eukaryotic genome complexity is enriched by embedded introns which expand the number of proteins produced by alternative splicing, provide unique environments to embed genes and regulatory elements, and create opportunities for new gene assembly through recombination and intron evolution. Such benefits come at a cost, however, as approximately 15% of human genetic disorders result from splicing errors associated with cis- (splicing substrate) mutations that alter gene-delimited splice patterns or trans- (spliceosome subunit) mutations that may impair general pre-mRNA splicing. A better understanding of spliceosome composition and the molecular basis of splice site selection will facilitate the diagnosis and, ultimately, the treatment or correction of splicing-related disorders. The contribution of spliceosome assembly to the mechanism of pre-mRNA splicing is the focus of our work.

Metazoan genes may contain dozens of intron/exon borders, some of which are used only in response to specific developmental or environmental cues. For many genes it is the stable recruitment of the U2 snRNP to the branchpoint region of the pre-mRNA that modulates splice site choice. Pre-mRNA branchpoint recognition is complex and even in the case unregulated transcripts, progresses through the sequential association of multiple splicing factors (e.g., SF1/BBP – U2AF65/Mud2p) and snRNPs (U1, U2, and U6).

While the basic pathway of spliceosome assembly is well conserved through evolution, Saccharomyces cerevisiae (henceforth yeast) lacks canonical SR splicing factor regulators found in metazoa and relies on more rigidly conserved pre-mRNA consensus elements to direct splice site choice. Yeast gene structure is also simpler, with few genes containing more than a single intron. Accordingly, yeast offers an excellent model to investigate the assembly and function of the basal splicing apparatus in the absence of complications resulting from complex gene organization and splicing. Currently, we using genetic and proteomic approaches to investigate the dynamics of pre-mRNA branchpoint selection in vitro and in living cells.

  • Martínez-Matías, N Chorna, N González-Crespo, S Villanueva, L Montes-Rodríguez, I Melendez-Aponte, LM Roche-Lima, A Carrasquillo-Carrión, K Santiago-Cartagena, E Rymond, BC Babu, M Stagljar, I Rodriguez-Medina, JR "Toward the discovery of biological functions associated with the mechanosensor Mtl1p of Saccharomyces cerevisiae via integrative multi-OMICs analysis." Scientific reports 11, 1 (2021): 7411. Details.Full text
  • Rivera-Robles, MJ Medina-Velázquez, J Asencio-Torres, GM González-Crespo, S Rymond, BC Rodríguez-Medina, J Dharmawardhane, S "Targeting Cdc42 with the anticancer compound MBQ-167 inhibits cell polarity and growth in the budding yeast S. cerevisiae." Small GTPases 11, 6 (2020): 430-440. Details.Full text
  • Vélez-Segarra, V González-Crespo, S Santiago-Cartagena, E Vázquez-Quiñones, LE Martínez-Matías, N Otero, Y Zayas, JJ Siaca, R Del Rosario, J Mejías, I Aponte, JJ Collazo, NC Lasso, FJ Snider, J Jessulat, M Aoki, H.Rymond, BC Babu, M Stagljar, I Rodriguez-Medina, JR "Protein Interactions of the Mechanosensory Proteins Wsc2 and Wsc3 for Stress Resistance in <i>Saccharomyces cerevisiae</i>." G3 (Bethesda, Md.) 10, 9 (2020): 3121-3135. Details.
  • Santiago-Cartagena, E González-Crespo, S Vélez, V Martínez, N.Snider, J Jessulat, M Aoki, H.Minic, Z Akamine, P Mejías, I Pérez, LM Rymond, BC Babu, M Stagljar, I Rodriguez-Medina, JR "Identification and Functional Testing of Novel Interacting Protein Partners for the Stress Sensors Wsc1p and Mid2p of <i>Saccharomyces cerevisiae</i>." G3 (Bethesda, Md.) 9, 4 (2019): 1085-1102. Details.
  • Santiago, E Akamine, P Snider, J Wong, V Jessulat, M Deineko, V Gagarinova, A Aoki, H.Minic, Z Phanse, S San Antonio, A Cubano, LA Rymond, BC Babu, M Stagljar, I Rodriguez-Medina, JR "Novel Interactome of Saccharomyces cerevisiae Myosin Type II Identified by a Modified Integrated Membrane Yeast Two-Hybrid (iMYTH) Screen." G3 (Bethesda, Md.) 6, 5 (2016): 1469-74. Details.
  • Banerjee, D McDaniel, PM Rymond, BC "Limited portability of G-patch domains in regulators of the Prp43 RNA helicase required for pre-mRNA splicing and ribosomal RNA maturation in Saccharomyces cerevisiae." Genetics 200, 1 (2015): 135-47. Details.Full text
  • Rymond, BC "The branchpoint binding protein: in and out of the spliceosome cycle." Advances in experimental medicine and biology 693, (2010): 123-41. Details.
  • Pandit, S.Paul, S.Zhang, L.Chen, M.Durbin, N.Harrison, SM Rymond, BC "Spp382p interacts with multiple yeast splicing factors, including possible regulators of Prp43 DExD/H-Box protein function." Genetics 183, 1 (2009): 195-206. Details.Full text
  • Wang, Q.Zhang, L.Lynn, B.Rymond, BC "A BBP-Mud2p heterodimer mediates branchpoint recognition and influences splicing substrate abundance in budding yeast." Nucleic acids research 36, 8 (2008): 2787-98. Details.Full text
  • Rymond, B. "Targeting the spliceosome." Nature chemical biology 3, 9 (2007): 533-5. Details.Full text
  • Pandit, S.Lynn, B.Rymond, BC "Inhibition of a spliceosome turnover pathway suppresses splicing defects." Proceedings of the National Academy of Sciences of the United States of America 103, 37 (2006): 13700-5. Details.Full text
  • Wang, Q.He, J.Lynn, B.Rymond, BC "Interactions of the yeast SF3b splicing factor." Molecular and cellular biology 25, 24 (2005): 10745-54. Details.Full text
  • Dembla-Rajpal, N.Seipelt, R.Wang, Q.Rymond, BC "Proteasome inhibition alters the transcription of multiple yeast genes." Biochimica et biophysica acta 1680, 1 (2004): 34-45. Details.Full text
  • Wang, Q.Rymond, BC "Rds3p is required for stable U2 snRNP recruitment to the splicing apparatus." Molecular and cellular biology 23, 20 (2003): 7339-49. Details.Full text
  • Vincent, K.Wang, Q.Jay, S.Hobbs, K.Rymond, BC "Genetic interactions with CLF1 identify additional pre-mRNA splicing factors and a link between activators of yeast vesicular transport and splicing." Genetics 164, 3 (2003): 895-907. Details.
  • Wang, Q.Hobbs, K.Lynn, B.Rymond, BC "The Clf1p splicing factor promotes spliceosome assembly through N-terminal tetratricopeptide repeat contacts." The Journal of biological chemistry 278, 10 (2003): 7875-83. Details.Full text
  • Chung, S.Zhou, Z.Huddleston, KA Harrison, DA Reed, R.Coleman, TA Rymond, BC "Crooked neck is a component of the human spliceosome and implicated in the splicing process." Biochimica et biophysica acta 1576, 3 (2002): 287-97. Details.Full text
  • Chung, S.McLean, MR Rymond, BC "Yeast ortholog of the Drosophila crooked neck protein promotes spliceosome assembly through stable U4/U6.U5 snRNP addition." RNA (New York, N.Y.) 5, 8 (1999): 1042-54. Details.Full text
  • Seipelt, RL Zheng, B.Asuru, A.Rymond, BC "U1 snRNA is cleaved by RNase III and processed through an Sm site-dependent pathway." Nucleic acids research 27, 2 (1999): 587-95. Details.Full text
  • Lybarger, S.Beickman, K.Brown, V.Dembla-Rajpal, N.Morey, K.Seipelt, R.Rymond, BC "Elevated levels of a U4/U6.U5 snRNP-associated protein, Spp381p, rescue a mutant defective in spliceosome maturation." Molecular and cellular biology 19, 1 (1999): 577-84. Details.Full text
  • Xie, J.Beickman, K.Otte, E.Rymond, BC "Progression through the spliceosome cycle requires Prp38p function for U4/U6 snRNA dissociation." The EMBO journal 17, 10 (1998): 2938-46. Details.Full text
  • McLean, MR Rymond, BC "Yeast pre-mRNA splicing requires a pair of U1 snRNP-associated tetratricopeptide repeat proteins." Molecular and cellular biology 18, 1 (1998): 353-60. Details.Full text
  • Roy, J.Zheng, B.Rymond, BC Woolford JL, Jr "Structurally related but functionally distinct yeast Sm D core small nuclear ribonucleoprotein particle proteins." Molecular and cellular biology 15, 1 (1995): 445-55. Details.Full text
  • Rodriguez-Medina, JR Rymond, BC "Prevalence and distribution of introns in non-ribosomal protein genes of yeast." Molecular & general genetics : MGG 243, 5 (1994): 532-9. Details.
  • Lockhart, SR Rymond, BC "Commitment of yeast pre-mRNA to the splicing pathway requires a novel U1 small nuclear ribonucleoprotein polypeptide, Prp39p." Molecular and cellular biology 14, 6 (1994): 3623-33. Details.Full text
  • Rymond, BC Rokeach, LA Hoch, SO "Human snRNP polypeptide D1 promotes pre-mRNA splicing in yeast and defines nonessential yeast Smd1p sequences." Nucleic acids research 21, 15 (1993): 3501-5. Details.Full text
  • Rymond, BC "Convergent transcripts of the yeast PRP38-SMD1 locus encode two essential splicing factors, including the D1 core polypeptide of small nuclear ribonucleoprotein particles." Proceedings of the National Academy of Sciences of the United States of America 90, 3 (1993): 848-52. Details.Full text
  • Blanton, S.Srinivasan, A.Rymond, BC "PRP38 encodes a yeast protein required for pre-mRNA splicing and maintenance of stable U6 small nuclear RNA levels." Molecular and cellular biology 12, 9 (1992): 3939-47. Details.Full text
  • Rymond, BC "Identification of sites of pre-MRNA/spliceosome association." SAAS bulletin, biochemistry and biotechnology 4, (1991): 76-80. Details.
  • Rymond, BC "The branchpoint binding protein: in and out of the spliceosome cycle." Advances in experimental medicine and biology 693, (0): 123-41. Details.

Rymond, BC., Going my way? A tale of enzyme recruitment and activation 2016, Atlas of Science,

Access options

Get full journal access for 1 year

All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.

Get time limited or full article access on ReadCube.

All prices are NET prices.


Differential detergent extraction was used to evaluate the extractability of kinesin in cultured cells. Brief extraction of cells with 0.015% digitonin creates holes in the plasma membrane with minimal effects on intracellular organelles (Mackall et al., 1979 Womack et al., 1983 Ramsby and Makowski, 1998). Cultured cells extracted under these conditions release 90–100% of traditional cytosolic markers such as lactate dehydrogenase and carbonic anhydrase, but mitochondrial, lysosomal, and endoplasmic reticulum markers are retained (Ramsby et al., 1994). The proteins released by digitonin treatment include myosin II, calpastatin and other proteins >300 kDa in molecular mass (Weigel et al., 1983 Ramsby et al., 1994). In contrast, extraction of cells with Triton X-100 solubilizes many plasma membrane and intracellular organelle membrane proteins in a time- and concentration-dependent manner (Ramsby and Makowski, 1998).

The behavior of kinesin during detergent extractions was compared with that of a protein known to be soluble in vivo. BHK21 cells constitutively expressing GFP were fixed directly or extracted before fixation with either Triton X-100 or digitonin (Figure1). When fixed without extraction GFP was retained in the cell, but even the mildest detergent treatments led to rapid loss of cytoplasmic GFP, leaving only a small residual fraction in nuclei. Comparison between GFP and kinesin distributions in unextracted cells suggested that these two proteins did not colocalize. GFP permeated the cell, matching well to cell boundaries and thickness, but kinesin immunoreactivity appeared to be more restricted, perhaps enriched in selected cellular domains.

Fig. 1. Soluble GFP but not kinesin is released from detergent-permeablilized cells. Fluorescent images of unextracted (A), 0.015% digitonin-extracted (B), and 0.1% Triton X-100-extracted (C) wild-type BHK21 cells (H2) or BHK21cells stably expressing GFP are shown. After fixation, cells were processed for immunofluorescence with a mouse anti-KHC antibody (H2) or GFP fluorescence was directly visualized (GFP green, left column). All cells were also stained with an anti-tubulin antibody (red) and with the nuclear marker To-Pro3 (blue middle column). Merged images are overlays of pseudocolored green–red–blue images (right column). Thin colored lines separate images of cells from different fields. The pattern of kinesin immunoreactivity differs from GFP fluorescence even in unextracted cells. These differences are more obvious after detergent extraction. Mild detergent extraction (digitonin) removes most of the GFP, except for a residual nuclear fraction. In contrast, significant kinesin immunoreactivity remains visible throughout the cell even after harsher detergent extraction (Triton X-100), indicating that most kinesin is not soluble.

Digitonin extraction before fixation revealed more striking differences between GFP and kinesin localization (Figure 1). Virtually all GFP was extracted from cytoplasmic domains within 4 min, leaving only a weak signal in the nucleus. In contrast, the bulk of the kinesin remained as discrete structures that were often closely apposed to microtubules in double-label studies. Significant punctate kinesin immunoreactivity remains even after more stringent extractions using Triton X-100 under conditions in which intracellular organelles begin to be extracted (Ramsby and Makowski, 1998). Although kinesin immunoreactivity appeared reduced with Triton X-100 treatment (Figure 1), much kinesin remained as punctate structures. Longer extractions and higher concentrations of Triton X-100 that disrupt internal membranes substantially reduced kinesin immunoreactivity ( Morfin et al., 2000Pfister et al., 1989b). Triton X-100 extraction also eliminated essentially all cytoplasmic GFP but was no more effective than digitonin in removing nuclear GFP. Although immunofluorescence methods are semiquantitative, they demonstrated that most kinesin immunoreactivity is resistant to treatments that release cytosolic proteins.

A more quantitative measure of kinesin bound to MBOs can be obtained by subcellular fractionation. Kinesin levels were evaluated by quantitative immunoblots of two purified organelle fractions (V1 and V2) and the supernatant (S) using standard cell fractionation methods. Consistent with previous reports (Hollenbeck, 1989), ∼70% of cellular kinesin was present in the supernatant with control buffers (Figure 2a). A wide range of buffer conditions and enzyme inhibitors were evaluated for an ability to affect the partitioning of kinesin between membrane and soluble fractions. Two treatments significantly increased the amount of kinesin in membrane fractions: NEM, a sulfhydryl modifying agent, and EDTA, a chelator of divalent cations. Both were effective at millimolar concentrations when added to homogenization buffers.

Fig. 2. Effects of NEM on kinesin release from vesicles during homogenization. (a) Microsomal vesicles were purified by homogenizing fresh bovine brains either with or without 2 mM NEM. Three fractions were defined: the 39,800 × gpellet (V1), the 260,000 × g pellet (V2), and the 260,000 × g supernatant (S). The supernatant and vesicle fractions were probed for the presence of kinesin using the H2 antibody on quantitative immunoblots as described previously (Pfister et al., 1989b). The presence of 2 mM NEM during homogenization minimized kinesin release from vesicles during homogenization. Error bars represent ±SEM n = 3. (b) The concentration dependence of NEM effects on the amount of kinesin in V2 was assayed by varying the concentrations of NEM added to homogenization buffer from 0.1 to 5.0 mM before the homogenization. Kinesin was quantitated as described above. NEM at concentrations of 1–2 mM maximally inhibited kinesin release from vesicles.

Addition of NEM to homogenization buffer before extraction alkylates any free sulfhydryls that become accessible during homogenization and inhibits enzyme activities requiring free sulfhydryls. Millimolar NEM in the homogenization buffer increased the amount of total protein in both V1 and V2 fractions only ∼5%. In contrast, the amount of kinesin associated with V1 and V2 membrane fractions increased approximately threefold, going from ∼0.5% to ∼1.5% of total protein. Under these conditions, >90% of recovered kinesin partitioned with vesicle fractions after NEM treatment (Figure 2), compared with 30% of total kinesin in vesicle fractions under standard conditions. Kinesin associated with MBOs after NEM treatment remained bound through sucrose gradient fractionation, indicating that increased membrane-bound kinesin with NEM treatment was not due to aggregation of soluble kinesin. Consistent with this, in vitro studies on inhibition of kinesin ATPase, microtubule binding and gliding assays by NEM, showed that NEM-treated kinesin does not aggregate but remains soluble (Pfister et al., 1989a Sickles et al., 1996). These experiments indicate that NEM inhibits kinesin release from MBOs during homogenization and suggested that at least one NEM-sensitive pathway in cytoplasmic extracts releases kinesin from MBOs during homogenization.

A comparable increase of kinesin in membrane fractions was produced by addition of 5 mM EDTA to homogenization buffers. In Figure3a, the upper histogram shows that NEM, EDTA, or the combination of NEM and EDTA treatments had little effect on the total amount of protein in cytosolic and sonicated membrane fractions. Similarly, none of these treatments altered the distribution of either hsc70, a predominantly cytoplasmic protein, or synaptophysin, an integral membrane protein (Figure 3b). However, the lower histogram shows that treatment with either NEM (51%) or EDTA (54%) more than doubled the amount of kinesin in membrane fractions compared with control buffers (26%). Adding both NEM and EDTA to homogenization buffer increased the amount of kinesin in membrane fractions to nearly 80% (Figure 3). The fact that NEM and EDTA effects on kinesin binding to membranes were at least partially additive suggests that multiple pathways may exist to release kinesin from MBOs and become activated during standard homogenization protocols.

Fig. 3. NEM and EDTA increase the amount of kinesin in membrane fractions after subcellular fractionation. (a) Bar graphs showing partitioning of total protein and kinesin in cytosolic versus membrane fractions. Changes in the partitioning of kinesin in the absence (control) or presence of NEM, EDTA, or NEM and EDTA combined are shown. Average kinesin levels were calculated from results of two quantitative immunoblots. Bars indicate the range of variation. (b) Representative immunoblots showing the partitioning of kinesin, hsc70, and the integral membrane protein p38 in control and treated samples. The partitioning of total protein, hsc70, and p38 between cytosolic and membrane fractions was unaltered by the various treatments. However, all treatments dramatically shifted kinesin from cytosolic to membrane fractions.

The ability of NEM and EDTA to inhibit release of kinesin from MBOs suggested an active process but did not identify the cellular components responsible. Immunochemical studies had implicated KLCs in kinesin binding to vesicles (Hirokawa et al., 1989 Stenoien and Brady, 1997), so the KLC primary sequence was examined for motifs that might be involved in regulation of membrane binding. The central domain of KLC contains five imperfect tandem repeats of 42 amino acids each (Cyr et al., 1991). These repeats have a high degree of similarity to each other and are conserved to an unusually high degree (>95% identity) across species (Brady, 1995 Stenoien and Brady, 1997). Our previous work had implicated these tandem repeat domains in kinesin binding to MBOs, because an antibody (KLC-ALL) directed against the repeat domain released kinesin from isolated vesicles in vitro and inhibited fast axonal transport. As a result, KLC tandem repeat domains were thought to mediate protein–protein or protein–lipid bilayer interactions needed for kinesin binding to MBOs (Stenoien and Brady, 1997).

Analysis of KLC sequences (Figure 4) revealed that tandem repeat domains contain conserved sequences characteristic of the “J-domain” motif first described in DnaJ (Tsai and Douglas, 1996). The predicted structure of the tandem repeat domains (Cyr et al., 1991) was also consistent with a J-domain function (Hill et al., 1995 Kelley, 1998). The hallmarks of a J-domain are a tripeptide HPD sequence between two alpha helical-rich stretches (Figure 4), creating a finger-like structure that can interact with members of the hsp70 chaperone family. A variety of proteins have been found to contain J-domains, several of which mediate processes involving protein–membrane interactions and molecular chaperones (Cyr et al., 1994 Kelley, 1998). This suggested that an hsp70 chaperone might bind to KLC. The action of hsp70 proteins on kinesin–MBO interactions was tested by evaluating the in vitro ability of hsc70 to release kinesin from purified vesicle fractions (Figure 5). Purified V2 vesicles were incubated with or without hsc70 for 30 min at 37°C. Only 5% of vesicle-associated kinesin was released into the supernatant by incubation with buffer alone. In contrast, ∼50% of bound kinesin was released from vesicle surfaces by in vitro incubations with hsc70 and ATP (Figure 5a). This is likely to be an underestimate of kinesin releasable by hsc70, because vesicle fractions used for these assays were purified by standard cell fractionation methods. Kinesin release by hsc70 during homogenization is likely to be more extensive, because much endogenous membrane-bound kinesin was lost during purification (Figures 2 and 3). The effect of ATP alone was also assayed, because ATP is required for hsc70 function. Approximately 12% of kinesin was released into the supernatant of the sample after incubation with buffer plus ATP in the absence of added hsc70 (Figure5). This ATP-dependent release probably resulted from hsc70 contamination of vesicle fractions, because a small amount of hsc70 was consistently detectable in immunoblots of the vesicle fractions (see Figure 3). Moreover, immunoblots with anti-hsc70 antibody showed release of endogenous hsc70 from samples only in the presence of added ATP (our unpublished data).

Fig. 4. Structure and sequence homologies between KLC tandem repeats and J-domains. Alignment of KLC tandem repeat sequences with a prokaryote J-domain sequence from Escherichia coli DnaJ and two eukaryotic J-domains from auxilin and Sec63P is shown (see DISCUSSION for more details). The invariant motifs are an essential HPD sequence (star) and the arrangement of short, flanking stretches of alpha helix (brackets under aligned sequences). The position of the alpha helical stretches is based on the nuclear magnetic resonance structure of the J-domains in DnaJ (Hillet al., 1995). Sequences in these helical stretches are more variable across family members, although some residues appear to be either conserved (letters under the sequence) or homologous (dots under sequence). Shading shows conserved residues in multiple J-domain sequences. This alignment shows that the primary sequences of tandem repeats 1–2 and 3–4 are consistent with a J-domain function.

Fig. 5. Release of endogenous kinesin from vesicles by Hsc70. V2 vesicles (purified without the use of NEM as described in the text) were resuspended and incubated at a concentration of 1 mg/ml total protein with or without hsc70 for 30 min at 37°C in release buffer (HB plus 75 mM KCl). Hsc70 was used at a concentration of 10 μg/ml for a molar ratio for hsc70:kinesin of 2:1. After incubation, V2 vesicles were recentrifuged at 260,000 × g, and then supernatants and pellets were probed for the presence of kinesin using the H2 antibody on quantitative immunoblots as described above. (a) Purified hsc70 released 50% of the endogenous kinesin remaining on V2 vesicles in a nucleotide-dependent manner. The reaction required Mg-ATP and was blocked by a nonhydrolyzable ATP analogue (AMP-PNP) and EDTA. The amount of kinesin released by hsc70 in this assay was comparable with that released by an antibody against the KLC tandem repeat domain (Stenoien and Brady, 1997). (b) When 2 mM NEM is added to vesicles in vitro at the same time as the hsc70 (time 0), hsc70-mediated release is blocked. The inhibition of hsc70-mediated kinesin release by NEM was eliminated by simultaneous addition of DTT (2 mM). The ability of NEM to inhibit hsc70-mediated release permitted definition of a time course for kinesin release. This could be estimated by adding NEM (2 mM) at the indicated times after the start of incubation, where the start of incubation is the addition of hsc70. When the reaction is allowed to proceed for 5 min before addition of NEM, substantial amounts of kinesin were released. Release of kinesin by hsc70 was essentially complete between 10 and 20 min, so addition of NEM at times >10 min had no effect on the amount of kinesin released by hsc70. (c) The target for NEM modification is a vesicle component, not hsc70 itself. To determine the site of action for NEM, the amount of kinesin remaining bound to vesicles (V) was compared with the amount of kinesin found in the soluble fraction (S) for five different conditions. When vesicles were pretreated with 5 mM NEM, the amount of kinesin released from vesicles by untreated hsc70 (VS pair 2) was similar to that seen without hsc70 (VS pair 1) or with NEM present during the hsc70 incubation (VS pair 3). In contrast, hsc70 pretreated with 5 mM NEM (VS pair 4) exhibited an ability to release kinesin comparable with that of untreated hsc70 (VS pair 5). For pretreatments with NEM, unreacted NEM was neutralized by addition of excess DTT before assay. Error bars represent ±SEM n = 3 experiments unless otherwise specified.

The roles of divalent cations and ATP hydrolysis in release of vesicle-bound kinesin by hsc70 were assessed because hsc70 function requires Mg ++ -dependent hydrolysis of ATP (Wilbanks et al., 1994). Addition of millimolar EDTA in the absence of added Mg ++ significantly reduced the amount of kinesin released by hsc70/ATP to ∼20% (Figure 5a). This was consistent with observations that EDTA increased the amount of kinesin retained by MBOs during homogenization (Figure 3). Under the conditions of the assay, EDTA did not completely inhibit, probably as a result of residual divalent cations present in the vesicle fraction and added ATP. Efficiency of release was also reduced by incubation with either AMP-PNP or adenosine 5′-[β,γ-methylene]triphosphate, two nonhydrolyzable analogues of ATP. Both ATP analogues reduced hsc70-mediated release of kinesin from vesicles to ∼30% (Figure 5a). Neither of these analogues bind tightly to hsc70 but act as weak competitive inhibitors in the presence of residual ATP from vesicle fractions. Together, these results indicate that binding and hydrolysis of MgATP is required for hsc70-mediated release of kinesin from MBOs.

Release of membrane-bound kinesin by hsc70 was also sensitive to NEM in vitro (Figure 5b). When 2 mM NEM and hsc70 were added at the same time (time 0), kinesin release by hsc70 was effectively blocked. This block required alkylation of a sulfhydryl by NEM because addition of comparable amounts of NEM inactivated by DTT at time 0 had no effect on release of kinesin by hsc70. However, addition of NEM to samples at various times after addition of hsc70 generated a time course for kinesin release (Figure 5b). Adding NEM >20 min after hsc70 did not affect the amount of kinesin released into supernatant, but clear decrements in kinesin release resulted if NEM was added ≤10 min after starting hsc70 incubation. If vesicle fractions were pretreated with 5 mM NEM and excess NEM was neutralized by addition of excess DTT before addition of hsc70, hsc70 was no longer able to release kinesin from vesicle fractions (Figure 5b). In contrast, a similar pretreatment of hsc70 with NEM had no effect on its ability to release kinesin from vesicles. Thus, the NEM-sensitive component required for hsc70 release of kinesin must be an MBO-associated component.

To characterize interactions between KLC and hsc70 further, fusion proteins containing different KLC constructs were added to the kinesin release assay. Four different bacterially expressed GST fusion proteins were used: 1) GST-LCA contains full-length KLC A fused to GST 2) GST-TR1 contains KLC tandem repeat amino acids 238–321 fused to GST 3) GST-TR2 contains KLC tandem repeat amino acids 238–488 fused to GST and 4) GST alone was used as a control in these assays. GST-LCA, GST-TR1, and GST-TR2 all inhibited release of kinesin by hsc70, but GST alone was ineffective (Figure 6a). GST-TR1 and GST-TR2 were more effective than GST-LCA in inhibiting kinesin release by hsc70 (Figure 6a). Differences in degree of inhibition between GST-LCA and GST-TR1/TR2 raised the possibility of negative cooperativity between tandem repeats and other KLC domains. When the abilities of GST-LCA and GST-TR1 to inhibit kinesin release was tested at various molar ratios (Figure 6b), both GST-LCA and GST-TR1 approached saturation at a 1:1 molecular ratio to hsc70. However, GST-TR1 was more effective at inhibiting kinesin release than GST-LCA at all molar ratios. GST-LCA never reduced kinesin release by hsc70 to less than half-maximal, whereas GST-TR1 almost completely inhibited hsc70-mediated kinesin release.

Fig. 6. Fusion proteins containing KLC or KLC tandem repeats inhibited the ability of hsc70 to release kinesin from vesicles. (a) Hsc70-mediated release of kinesin from V2 vesicle fractions was assayed as described above. Four different constructs were generated: GST, GST-LCA, GST-TR1, and GST-TR2. At a molar ratio of 1:1 for fusion protein:hsc70, GST-LCA, GST-TR1, and GST-TR2 constructs inhibited release of kinesin by hsc70. In contrast, GST alone was inactive in these assays. Constructs containing only KLC tandem repeat domains (TR1 and TR2) were more effective than ones also containing other KLC domains (GST-LCA). Recombinant KLC and tandem repeat fusion proteins differ in their relative efficacy to inhibit hsc70-mediated release of kinesin. (b) The lesser ability of GST-LCA to inhibit hsc70-mediated release relative to GST-TR1/TR2 might reflect differences in the amount of active protein or an effect of domains in KLC other than the tandem repeats. If the amount of active protein differs, increased molar ratios of KLC to hsc70 should reduce observed differences. However, at all molar ratios tested, GST-TR1 was more effective at inhibiting kinesin release by hsc70 than GST-LCA. The effects of GST-LCA appeared to plateau at ∼30%. (c) To determine whether GST-LCA and GST-TR1 differed in their interactions with hsc70, the ability of different constructs to activate hsc70 ATPase activity was evaluated. Both GST-LCA and GST-TR1 stimulated hsc70 ATPase activity to a similar extent, indicating a comparable ability to bind and activate hsc70 ATPase. GST alone was inactive. Therefore, differences in the ability of LCA and TR1 fusion proteins to inhibit hsc70-mediated release of kinesin from vesicles were not due to differences in their ability to interact with hsc70, so KLC domains outside the tandem repeats must affect the ability of hsc70 to release kinesin from MBOs. Error bars represent ±SEM n = 3 experiments unless otherwise specified.

Conformational differences between GST-LCA and GST-TR1/TR2 that affected interactions with hsc70 could also explain this difference. The ability of a J-domain to bind hsc70 can be evaluated by assaying its stimulation of hsc70 ATPase activity (Cyret al., 1994 Tsai and Douglas, 1996 Greene et al., 1998). GST alone was inactive in hsc70 ATPase assays, but both GST-LCA and GST-TR1 stimulated hsc70 ATPase activity to a similar extent (Figure 6c). This indicates that tandem repeats and full-length KLC fusion proteins were comparable in their ability to bind and activate hsc70 ATPase. Taken together, these results suggest that KLC J-domain motifs interact directly with hsc70. Differences between KLC and tandem repeat domain inhibition of hsc70-mediated kinesin release from vesicles may reflect interaction of other domains in KLC with membrane components.

Finally, the ability of exogenous hsc70 to release kinesin from membranes in situ was examined (Figure7). Cells were permeabilized by incubation with 0.01% Triton X-100 for 4 min in the presence or absence of exogenous hsc70 before fixation. Brief incubation with hsc70 produced a consistent reduction in kinesin immunoreactivity (Figure 7, a and b). Kinesin immunoreactivity in processes was most severely affected, but there was also depletion of kinesin in cell bodies relative to control cells. The fact that not all kinesin was extracted by a short hsc70 incubation is consistent with the biochemical studies (see Figure 5) but may also represent either a fraction of kinesin that is resistant to hsc70 release or one that requires an additional cofactor not preserved under these conditions.

Fig. 7. Exogenous hsc70 releases kinesin from gently permeabilized cells. Kinesin immunofluorescence in untreated (A and B) and hsc70-treated (C and D) cells is shown. Images A and C were acquired with a 40×, objective and B and D were acquired with a 63× objective calibration bars are 20 μm in each case. Insets in B and D were digitally enlarged (4×) from the areas demarcated by black boxes. Kinesin inmunoreactivity declined after hsc70 treatment. Comparing A and C, the amount of kinesin in processes and in the perinuclear halo is reduced. Comparing B and D, this erosion of kinesin immunoreactivity in the perinuclear halo is more readily visulalized. The effect is most obvious near the cell center (B and D, insets), where the density of punctuate structures is reduced, resulting in a thinner perinuclear stain. Longer treatments will remove more kinesin immunoreactivity.

A single vesicular glutamate transporter is sufficient to fill a synaptic vesicle

Quantal size is the postsynaptic response to the release of a single synaptic vesicle and is determined in part by the amount of transmitter within that vesicle. At glutamatergic synapses, the vesicular glutamate transporter (VGLUT) fills vesicles with glutamate. While elevated VGLUT expression increases quantal size, the minimum number of transporters required to fill a vesicle is unknown. In Drosophila DVGLUT mutants, reduced transporter levels lead to a dose-dependent reduction in the frequency of spontaneous quantal release with no change in quantal size. Quantal frequency is not limited by vesicle number or impaired exocytosis. This suggests that a single functional unit of transporter is both necessary and sufficient to fill a vesicle to completion and that vesicles without DVGLUT are empty. Consistent with the presence of empty vesicles, at dvglut mutant synapses synaptic vesicles are smaller, suggesting that vesicle filling and/or transporter level is an important determinant of vesicle size.


Figure 1. DVGLUT Protein Levels and Staining…

Figure 1. DVGLUT Protein Levels and Staining Are Reduced in dvglut Mutants

Figure 2. mEJP Frequency but Not mEJP…

Figure 2. mEJP Frequency but Not mEJP Amplitude Is Decreased in dvglut Mutants

Figure 3. No Spontaneous Events Are Detected…

Figure 3. No Spontaneous Events Are Detected in dvglut Null Mutants Despite Normal Glutamate Responses

Figure 4. Synaptic Vesicles Are Abundant but…

Figure 4. Synaptic Vesicles Are Abundant but Smaller on Average in dvglut Mutants

Figure 5. Cycling Vesicle Pool Is Unchanged…

Figure 5. Cycling Vesicle Pool Is Unchanged in dvglut Hypomorphs, Despite Decreased EJP Amplitude