TY - JOUR
T1 - Active translocon complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum
AU - Nikonov, Andrei V.
AU - Snapp, Erik
AU - Lippincott-Schwartz, Jennifer
AU - Kreibich, Gert
PY - 2002
Y1 - 2002
N2 - 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.
AB - 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.
KW - Endoplasmic reticulum
KW - FRAP
KW - Lateral mobility
KW - Oligosaccharyltransferase
KW - Translocon complex
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UR - http://www.scopus.com/inward/citedby.url?scp=0036327783&partnerID=8YFLogxK
U2 - 10.1083/jcb.200201116
DO - 10.1083/jcb.200201116
M3 - Article
C2 - 12163472
AN - SCOPUS:0036327783
SN - 0021-9525
VL - 158
SP - 497
EP - 506
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 3
ER -