Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro

J Cell Biol. 1993 Feb;120(4):865-75. doi: 10.1083/jcb.120.4.865.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Consensus Sequence
  • Endoplasmic Reticulum / metabolism*
  • Endoplasmic Reticulum / physiology*
  • Fungal Proteins / metabolism*
  • Genes, Fungal
  • Macromolecular Substances
  • Membrane Proteins / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Nuclear Pore Complex Proteins
  • Organelles / metabolism*
  • Restriction Mapping
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins
  • Sequence Alignment

Substances

  • Fungal Proteins
  • Macromolecular Substances
  • Membrane Proteins
  • Nuclear Pore Complex Proteins
  • SEC13 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins

Associated data

  • GENBANK/L05929