Active translocon complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum.
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 (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.
Nikonov
Andrei V
AV
Department of Cell Biology, New York University School of Medicine, New York 10016, USA.
Snapp
Erik
E
Lippincott-Schwartz
Jennifer
J
Kreibich
Gert
G
D000818:Animals; D002478:Cells, Cultured; D002999:Clone Cells / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D004058:Diffusion; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005057:Eukaryotic Cells / Q000378:metabolism* / Q000648:ultrastructure; D005786:Gene Expression Regulation / Q000502:physiology; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006602:Hexosyltransferases; D007202:Indicators and Reagents; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism*; D014176:Protein Biosynthesis / Q000502:physiology*; D021381:Protein Transport / Q000502:physiology*; D011506:Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000069816:SEC Translocation Channels; D013997:Time Factors; D014166:Transferases / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000069816:SEC Translocation Channels; D016159:Tumor Suppressor Protein p53; D049452:Green Fluorescent Proteins; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase