Translocation of a long amino-terminal domain through ER membrane by following signal-anchor sequence.
Type I signal-anchor sequences mediate translocation of the N-terminal domain (N-domain) across the endoplasmic reticulum (ER) membrane. To examine the translocation in detail, dihydrofolate reductase (DHFR) was fused to the N-terminus of synaptotagmin II as a long N-domain. Translocation was arrested by the DHFR ligand methotrexate, which stabilizes the folding of the DHFR domain, and resumed after depletion of methotrexate. The targeting of the ribosome-nascent chain complex to the ER requires GTP, whereas N-domain translocation does not require any nucleotide triphosphates. Significant translocation was observed even in the absence of a lumenal hsp70 (BiP). When the nascent polypeptide was released from the ribosomes after the membrane targeting, the N-domain translocation was suppressed and the nascent chain was released from the translocon. Ribosomes have a crucial role in maintaining the translocation-intermediate state. The translocation of the DHFR domain was greatly impaired when it was separated from the signal-anchor sequence. Unfolding and translocation of the DHFR domain must be driven by the stroke of the signal-anchor sequence into translocon.
Kida
Yuichiro
Y
CREST of the Japan Science and Technology Agency, University of Hyogo, Ako, Hyogo, Japan.
Mihara
Katsuyoshi
K
Sakaguchi
Masao
M
D000123:Acetyltransferases; D000255:Adenosine Triphosphate / Q000494:pharmacology; D051046:Amino-Acid N-Acetyltransferase; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects; D011691:Puromycin / Q000494:pharmacology; D051381:Rats; D012270:Ribosomes / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D021382:Protein Sorting Signals; D011691:Puromycin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D013762:Tetrahydrofolate Dehydrogenase; D000123:Acetyltransferases; D051046:Amino-Acid N-Acetyltransferase; C494877:Nags protein, mouse; D008727:Methotrexate