Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase.
The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 microm CuSO(4)) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.
Liu
Jingxuan
J
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058, USA.
Sitaram
Anand
A
Burd
Christopher G
CG
D027682:Cation Transport Proteins / Q000378:metabolism*; D003300:Copper / Q000502:physiology*; D000079923:Copper Transporter 1; D004705:Endocytosis / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology*; D043743:Ubiquitin-Protein Ligase Complexes / Q000502:physiology*; D014617:Vacuoles / Q000201:enzymology / Q000378:metabolism*
C490078:CTR1 protein, S cerevisiae; D027682:Cation Transport Proteins; D000079923:Copper Transporter 1; D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D043743:Ubiquitin-Protein Ligase Complexes; C469287:RSP5 protein, S cerevisiae