Detection of a novel 9-kDa endoplasmic reticulum membrane protein in mammalian cells by chemical cross-linking with translocating nascent peptides.
It is widely accepted that a proteinous channel participates in the co-translational translocation of proteins across the endoplasmic reticulum (ER) membrane. Truncated mRNAs encoding N-terminal 70, 75, 88, and 110 amino acids of interleukin 2 were translated with wheat germ cell-free system in the presence of rough microsomal membrane (RM), and integral membrane proteins were probed with the translocating nascent peptides by using a cross-linking reagent (DSS). Two membrane proteins, 9 kDa (Cp9, cross-linking partner 9 k) and 39 kDa (Cp39, cross-linking partner 39 k), were cross-linked with the 75 amino acids nascent peptide. When NEM-treated RM was used for the translocation reaction, neither Cp9 nor Cp39 proteins were cross-linked. When the translation products were treated with puromycin before the cross-linking, both proteins were not cross-linked. The cross-linked products of Cp9 and Cp39 were not extracted by alkaline extraction of the membrane, not sensitive to endoglycosidase H, and did not bind to Con A-Sepharose. These results indicate that both of the cross-linking partners were nonglycosylated integral membrane proteins. Cp39 was cross-linked with the 70, 88, and 110 amino acid nascent peptides as well as the 75 amino acid peptide, whereas Cp9 reacted only with the nascent peptides consisting of 70 and 75 amino acid residues. Even after the digitonin treatment of the RM with the translocating intermediates, the cross-linked products with Cp9 and Cp39 were detected. Cp9 and Cp39 seem to be tightly associated with the ribosome-nascent peptide complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Kuroiwa
T
T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka.
Sakaguchi
M
M
Mihara
K
K
Omura
T
T
D000818:Animals; D001692:Biological Transport; D002474:Cell-Free System; D003432:Cross-Linking Reagents; D004072:Digitonin / Q000494:pharmacology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D005033:Ethylmaleimide / Q000494:pharmacology; D006031:Glycosylation; D006596:Hexosaminidases / Q000494:pharmacology; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism*; D007425:Intracellular Membranes / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D013388:Succinimides
D003432:Cross-Linking Reagents; D007376:Interleukin-2; D008565:Membrane Proteins; D012333:RNA, Messenger; D013388:Succinimides; D006596:Hexosaminidases; D004072:Digitonin; D005033:Ethylmaleimide; C019358:disuccinimidyl suberate