Structural and functional dissection of a membrane glycoprotein required for vesicle budding from the endoplasmic reticulum.
Sec12p is a membrane glycoprotein required for the formation of a vesicular intermediate in protein transport from the endoplasmic reticulum to the Golgi apparatus in Saccharomyces cerevisiae. Comparison of the N-linked glycosylation of Sec12p, a Sec12p-invertase hybrid protein, and a derivative of Sec12p lacking 71 carboxy-terminal amino acids showed that Sec12p is a type II membrane protein. Analysis of two truncated forms of Sec12p and of a temperature-sensitive mutant indicated that the C-terminal domain of Sec12p is not essential for protein transport, whereas the integrity and membrane attachment of the cytoplasmic N-terminal domain are essential. Expression of a soluble cytoplasmic domain dramatically inhibited the growth of a sec12 temperature-sensitive strain by increasing the transport defect at a normally permissive temperature. This growth inhibition as well as the sec12 temperature-sensitive defect were suppressed by the overproduction of Sar1p, a small GTP-binding protein that participates in protein transport. Sar1p membrane association was enhanced by elevated levels of Sec12p. These results suggest that the cytoplasmic domain of Sec12p interacts with Sar1p and that the complex may function to promote vesicle formation.
d'Enfert
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C
Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of California, Berkeley 94720.
Barlowe
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C
Nishikawa
S
S
Nakano
A
A
Schekman
R
R
D001483:Base Sequence; D002872:Chromosome Deletion; D004271:DNA, Fungal / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D005838:Genotype; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014415:Tunicamycin / Q000494:pharmacology
D004271:DNA, Fungal; D005656:Fungal Proteins; D020662:Guanine Nucleotide Exchange Factors; D008562:Membrane Glycoproteins; D009841:Oligonucleotides; C071239:SEC12 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014415:Tunicamycin