Active translocon complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum

Andrei V. Nikonov, Erik Snapp, Jennifer Lippincott-Schwartz, Gert Kreibich

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyl-transferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP-Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP-Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP-Dad1 in the ER membranes, but to a level that is still lower than that of free GFP-Dad1. This suggests that GFP-Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains.

Original languageEnglish (US)
Pages (from-to)497-506
Number of pages10
JournalJournal of Cell Biology
Volume158
Issue number3
DOIs
StatePublished - 2002
Externally publishedYes

Fingerprint

Polyribosomes
Transferases
Endoplasmic Reticulum
Glycosylation
Translational Protein Modification
Membranes
Ribosomes
Membrane Proteins
Phenotype
Peptides
Temperature
Proteins

Keywords

  • Endoplasmic reticulum
  • FRAP
  • Lateral mobility
  • Oligosaccharyltransferase
  • Translocon complex

ASJC Scopus subject areas

  • Cell Biology

Cite this

Active translocon complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum. / Nikonov, Andrei V.; Snapp, Erik; Lippincott-Schwartz, Jennifer; Kreibich, Gert.

In: Journal of Cell Biology, Vol. 158, No. 3, 2002, p. 497-506.

Research output: Contribution to journalArticle

Nikonov, Andrei V. ; Snapp, Erik ; Lippincott-Schwartz, Jennifer ; Kreibich, Gert. / Active translocon complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum. In: Journal of Cell Biology. 2002 ; Vol. 158, No. 3. pp. 497-506.
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