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© The Rockefeller University Press, 0021-9525/2002/8/497 $5.00
The Journal of Cell Biology, Volume 158, Number 3, August 5, 2002 497-506

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

¹ Department of Cell Biology, New York University School of Medicine, New York, NY 10016
² Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, NY 10016
³ Unit of Organelle Biology, Cell Biology and Metabolism Branch, National Institute of Child Health & Human Development/National Institutes of Health, Bethesda, MD 20892

Address correspondence to Gert Kreibich, Department of Cell Biology, New York University School of Medicine, New York, NY 10016. Tel.: (212) 263-5317. Fax: (212) 263-8139. E-mail: kreibg01{at}popmail.med.nyu.edu

In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (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 (D_eff) 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.

Key Words: translocon complex; oligosaccharyltransferase; lateral mobility; FRAP; endoplasmic reticulum

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