Protein unfolding is not a prerequisite for endoplasmic reticulum-to-cytosol dislocation.
We examined the effects of protein folding on endoplasmic reticulum (ER)-to-cytosol transport (dislocation) by exploiting the well-characterized dihydrofolate reductase (DHFR) domain. DHFR retains the capacity to bind folate analogues in the lumen of microsomes and in the ER of intact cells, upon which it acquires a conformation resistant to proteinase K digestion. Here we show that a Class I major histocompatibility complex heavy chain fused to DHFR is still recognized by the human cytomegalovirus-encoded glycoproteins US2 and US11, resulting in dislocation of the fusion protein from the ER in vitro and in vivo. A folded state of the DHFR domain does not impair dislocation of Class I MHC heavy chains in vitro or in living cells. In fact, a slight acceleration of the dislocation of DHFR heavy chain fusion was observed in vitro in the presence of a folate analogue. These results suggest that one or more of the channels used for dislocation can accommodate polypeptides that contain a tightly folded domain of considerable size. Our data raise the possibility that the Sec61 channel can be modified to accommodate a folded DHFR domain for dislocation, but not for translocation into the ER, or that a channel altogether distinct from Sec61 is used for dislocation.
Tirosh
Boaz
B
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Furman
Margo H
MH
Tortorella
Domenico
D
Ploegh
Hidde L
HL
D003600:Cytosol / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D019286:Endopeptidase K / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005492:Folic Acid / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000069816:SEC Translocation Channels; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D008565:Membrane Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D005492:Folic Acid; D019286:Endopeptidase K