Role for adenosine triphosphate in regulating the assembly and transport of vesicular stomatitis virus G protein trimers.
We have characterized the process by which the vesicular stomatitis virus (VSV) G protein acquires its final oligomeric structure using density-gradient centrifugation in mildly acidic sucrose gradients. The mature wild-type VSV G protein is a noncovalently associated trimer. Trimers are assembled from newly synthesized G monomers with a t1/2 of 6-8 min. To localize the site of trimerization and to correlate trimer formation with steps in transport between the endoplasmic reticulum (ER) and Golgi complex, we examined the kinetics of assembly of the temperature-sensitive mutant VSV strain, ts045. At the nonpermissive temperature (39 degrees C), ts045 G protein is not transported from the ER. The phenotypic defect that inhibited export from the ER at the nonpermissive temperature was found to be the accumulation of ts045 G protein in an aggregate. After being shifted to the permissive temperature (32 degrees C), the ts045 G protein aggregate rapidly dissociated (t1/2 less than 1 min) to monomeric G protein which subsequently trimerized with the same kinetics as the wild-type G protein. Only trimers were transported to the Golgi complex. Kinetic studies, as well as the finding that trimerization occurred under conditions which block ER to Golgi transport (at both 15 and 4 degrees C), showed that trimers were formed in the ER. Depletion of cellular ATP inhibited both the dissociation of the aggregated intermediate of ts045 G protein as well as the formation of stable trimers. The results indicate that oligomerization of G protein occurs in several steps, is sensitive to cellular ATP, and is required for transport from the ER.
Doms
R W
RW
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Keller
D S
DS
Helenius
A
A
Balch
W E
WE
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D007700:Kinetics; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000378:metabolism
C028391:G protein, vesicular stomatitis virus; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; D000255:Adenosine Triphosphate