Quality control in the endoplasmic reticulum: folding and misfolding of vesicular stomatitis virus G protein in cells and in vitro.
Parallel experiments in living cells and in vitro were undertaken to characterize the mechanism by which misfolded and unassembled glycoproteins are retained in the ER. A thermoreversible folding mutant of vesicular stomatitis virus (VSV) G protein called ts045 was analyzed. At 39 degrees C, newly synthesized G failed to fold correctly according to several criteria: intrachain disulfide bonds were incomplete; the B2 epitope was absent; and the protein was associated with immunoglobulin heavy chain binding protein (BiP), a heat shock-related, ER protein. When the temperature was lowered to 32 degrees C, these properties were reversed, and the protein was transported to the cell surface. Upon the shift up from 32 degrees C back to 39 degrees C, G protein in the ER returned to the misfolded form and was retained, while the protein that had reached a pre-Golgi compartment or beyond was thermostable and remained transport competent. The misfolding reaction could be reconstituted in a cell free system using ts045 virus particles and protein extracts from microsomes. Taken together, the results showed that ER is unique among the organelles of the secretory pathway in containing specific factors capable of misfolding G protein at the nonpermissive temperature and thus participating in its retention.
de Silva
A M
AM
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.
Balch
W E
WE
Helenius
A
A
D000818:Animals; D020126:Brefeldin A; D002460:Cell Line; D002474:Cell-Free System; D003517:Cyclopentanes / Q000494:pharmacology; D004220:Disulfides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000939:Epitopes / Q000378:metabolism; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins; D008861:Microsomes / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013696:Temperature; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism*
D003517:Cyclopentanes; D004220:Disulfides; D000939:Epitopes; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D020126:Brefeldin A