Genome-wide analysis of AP-3-dependent protein transport in yeast.
The evolutionarily conserved adaptor protein-3 (AP-3) complex mediates cargo-selective transport to lysosomes and lysosome-related organelles. To identify proteins that function in AP-3-mediated transport, we performed a genome-wide screen in Saccharomyces cerevisiae for defects in the vacuolar maturation of alkaline phosphatase (ALP), a cargo of the AP-3 pathway. Forty-nine gene deletion strains were identified that accumulated precursor ALP, many with established defects in vacuolar protein transport. Maturation of a vacuolar membrane protein delivered via a separate, clathrin-dependent pathway, was affected in all strains except those with deletions of YCK3, encoding a vacuolar type I casein kinase; SVP26, encoding an endoplasmic reticulum (ER) export receptor for ALP; and AP-3 subunit genes. Subcellular fractionation and fluorescence microscopy revealed ALP transport defects in yck3Delta cells. Characterization of svp26Delta cells revealed a role for Svp26p in ER export of only a subset of type II membrane proteins. Finally, ALP maturation kinetics in vac8Delta and vac17Delta cells suggests that vacuole inheritance is important for rapid generation of proteolytically active vacuolar compartments in daughter cells. We propose that the cargo-selective nature of the AP-3 pathway in yeast is achieved by AP-3 and Yck3p functioning in concert with machinery shared by other vacuolar transport pathways.
Anand
Vikram C
VC
Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.
Daboussi
Lydia
L
Lorenz
Todd C
TC
Payne
Gregory S
GS
D033963:Adaptor Protein Complex 3 / Q000235:genetics / Q000502:physiology*; D000469:Alkaline Phosphatase / Q000032:analysis / Q000378:metabolism*; D047389:Casein Kinase I / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017353:Gene Deletion; D016681:Genome, Fungal; D049452:Green Fluorescent Proteins / Q000032:analysis; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D021122:Protein Subunits / Q000235:genetics; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins
D033963:Adaptor Protein Complex 3; D008565:Membrane Proteins; D021122:Protein Subunits; C503875:SVP26 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D047389:Casein Kinase I; C479667:YCK3 protein, S cerevisiae; D000469:Alkaline Phosphatase