Translocation of a long amino-terminal domain through ER membrane by following signal-anchor sequence

EMBO J. 2005 Sep 21;24(18):3202-13. doi: 10.1038/sj.emboj.7600788. Epub 2005 Aug 18.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyltransferases
  • Adenosine Triphosphate / pharmacology
  • Amino-Acid N-Acetyltransferase
  • Animals
  • COS Cells
  • Chlorocebus aethiops
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / enzymology
  • Endoplasmic Reticulum / metabolism*
  • Guanosine Triphosphate / pharmacology
  • HSP70 Heat-Shock Proteins / metabolism
  • Intracellular Membranes / drug effects
  • Intracellular Membranes / enzymology
  • Intracellular Membranes / metabolism*
  • Methotrexate / metabolism
  • Mice
  • Molecular Sequence Data
  • Protein Folding
  • Protein Sorting Signals / physiology*
  • Protein Transport / drug effects
  • Puromycin / pharmacology
  • Rats
  • Ribosomes / metabolism
  • Tetrahydrofolate Dehydrogenase / chemistry*
  • Tetrahydrofolate Dehydrogenase / metabolism*

Substances

  • HSP70 Heat-Shock Proteins
  • Protein Sorting Signals
  • Puromycin
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • Tetrahydrofolate Dehydrogenase
  • Acetyltransferases
  • Amino-Acid N-Acetyltransferase
  • Nags protein, mouse
  • Methotrexate