Discussion 4: Transport vesicle sorting

Paper

W. E. Balch, J. M. McCaffery, H. Plutner, M. G. Farquhar. 1994. Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell 76:841-852

Review:

Aridor, M., and W. E. Balch, 1996, Principles of selective transport: coat complexes hold the key. Trends Cell Biol. 6:315-320.

Main Point:

This paper presents evidence that contradicts the "bulk flow" model, and suggests that the VSV-G protein is concentrated in transport vesicles leaving the ER for the Golgi apparatus. The degree of concentration is quantitated in careful immunoEM studies. This finding indicates that transport of proteins from the ER may be selective, and models for such a selective process are discussed.

Notes:

  1. The vesicular stomatitis virus (VSV) G protein is the glycoprotein that forms the surface spikes in the virion (like the HA protein from influenza virus). "G" stands for glycoprotein. The VSV-G protein is synthesized on membrane-bound ribosomes in the ER, is inserted into the ER membrane, and is transported to the Golgi apparatus during passage to the cell surface, where the virion is assembled. VSV-G spans the membrane once, with a large amino terminal domain in the lumen, and a short C-terminal tail domain exposed to the cytoplasm.
  2. Digitonin used under the correct conditions selectively solubilizes membranes with a high cholesterol content, such as the plasma membrane, but leaves other cellular membranes intact.
  3. VTCs are vesicular-tubular clusters, which are extensions of the ER that participate in transport to the Golgi apparatus. VTCs are part of the cis Golgi reticulum, which is also called the "intermediate compartment."
  4. The thickness of sections prepared for EM are approximately the same as the diameter of transport vesicles and VTCs, which is significant in interpreting the serial section data.

Questions for Discussion:

  1. What model/hypothesis is examined by the authors?
  2. What experimental system is used to collect data on VSV-G transport? What is the significance of using ts045 VSV-G?
  3. What is the significance of the appearance of the coat on the transport vesicles?
  4. Why was calreticulin (CaBP3) immunolocalized in this study? Why was VSV-G immunolocalized with antibody VG1?
  5. What mechanism(s) might account for the "positive sorting" of VSV-G?