Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro.
The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane. SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins. The product of this gene, Sec13p, is a cytosolic protein peripherally associated with membranes. We developed a cell-free Sec13p-dependent vesicle formation reaction. Sec13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a Sec13p-dihydrofolate reductase fusion protein, respectively. These fractions were unable to support vesicle formation from the ER unless cytosol containing Sec13p was added. Cytosolic Sec13p fractionated by gel filtration as a large complex of about 700 kD. Fractions containing the Sec13p complex restored activity to the Sec13p- dependent vesicle formation reaction. Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of Sec13p, suggesting that another member of the complex becomes limiting when Sec13p is overproduced. Overproduced, monomeric Sec13p was inactive in the Sec13p-dependent vesicle formation assay.
Pryer
N K
NK
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Salama
N R
NR
Schekman
R
R
Kaiser
C A
CA
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000502:physiology*; D005656:Fungal Proteins / Q000378:metabolism*; D005800:Genes, Fungal; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D028861:Nuclear Pore Complex Proteins; D015388:Organelles / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment
D005656:Fungal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; C079620:SEC13 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins