Different transmembrane domains associate with distinct endoplasmic reticulum components during membrane integration of a polytopic protein.
We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the alpha and beta subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61alpha and Sec61beta during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the "stage" of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.
Meacock
Suzanna L
SL
School of Biological Sciences, University of Manchester, Manchester, M13 9PT United Kingdom.
Lecomte
Fabienne J L
FJ
Crawshaw
Samuel G
SG
High
Stephen
S
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009496:Neurotensin / Q000737:chemistry; D010179:Pancreas / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D017299:Rod Opsins / Q000737:chemistry; D000069816:SEC Translocation Channels; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D017299:Rod Opsins; D000069816:SEC Translocation Channels; D009496:Neurotensin