Assembly of the yeast vacuolar H+-ATPase occurs in the endoplasmic reticulum and requires a Vma12p/Vma22p assembly complex

J Cell Biol. 1998 Jul 13;142(1):39-49. doi: 10.1083/jcb.142.1.39.

Abstract

Three previously identified genes from Saccharomyces cerevisiae, VMA12, VMA21, and VMA22, encode proteins localized to the endoplasmic reticulum (ER). These three proteins are required for the biogenesis of a functional vacuolar ATPase (V-ATPase), but are not part of the final enzyme complex. Subcellular fractionation and chemical cross-linking studies have revealed that Vma12p and Vma22p form a stable membrane associated complex. Cross-linking analysis also revealed a direct physical interaction between the Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase. The interaction of the Vma12p/Vma22p complex with Vph1p was transient (half-life of approximately 5 min), reflecting trafficking of this V-ATPase subunit through the ER en route to the vacuolar membrane. Analysis of these protein-protein interactions in ER-blocked sec12 mutant cells indicated that the Vph1p-Vma12p/Vma22p interactions are quite stable when transport of the V-ATPase out of the ER is blocked. Fractionation of solubilized membrane proteins on a density gradient revealed comigration of Vma22p and Vma12p, indicating that they form a complex even in the absence of cross-linker. Vma12p and Vma22p migrated to fractions separate from Vma21p. Loss of Vph1p caused the Vma12p/Vma22p complex to sediment to less dense fractions, consistent with association of Vma12p/ Vma22p with nascent Vph1p in ER membranes. This is the first evidence for a dedicated assembly complex in the ER required for the assembly of an integral membrane protein complex (V-ATPase) as it is transported through the secretory pathway.

Publication types

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

MeSH terms

  • Endoplasmic Reticulum / metabolism*
  • Fungal Proteins / metabolism*
  • Intracellular Membranes / metabolism
  • Membrane Proteins / metabolism*
  • Molecular Chaperones
  • Proton-Translocating ATPases / biosynthesis*
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae Proteins*
  • Vacuolar Proton-Translocating ATPases*

Substances

  • Fungal Proteins
  • Membrane Proteins
  • Molecular Chaperones
  • Saccharomyces cerevisiae Proteins
  • VMA21 protein, S cerevisiae
  • VMA22 protein, S cerevisiae
  • VPH2 protein, S cerevisiae
  • Vacuolar Proton-Translocating ATPases
  • Proton-Translocating ATPases