Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole

J Cell Biol. 2001 Apr 16;153(2):381-96. doi: 10.1083/jcb.153.2.381.

Abstract

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

Publication types

  • 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

  • Aminopeptidases / genetics
  • Aminopeptidases / metabolism
  • Biological Transport / physiology*
  • Blotting, Western
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Fractionation
  • Cell Membrane / metabolism
  • Cytosol / metabolism
  • Glucose / metabolism
  • Humans
  • Microscopy, Fluorescence
  • Peroxisomes / metabolism
  • Pichia / genetics
  • Pichia / metabolism
  • Pichia / ultrastructure
  • Plasmids / genetics
  • Plasmids / metabolism
  • Protein Binding
  • Protein Precursors / genetics
  • Protein Precursors / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / physiology*
  • Saccharomyces cerevisiae / ultrastructure
  • Saccharomyces cerevisiae Proteins*
  • Signal Transduction / physiology*
  • Transport Vesicles / metabolism*
  • Vacuoles / metabolism*

Substances

  • Carrier Proteins
  • Protein Precursors
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Aminopeptidases
  • APE1 protein, S cerevisiae
  • Glucose