Systematic Mutational Analysis of the Intracellular Regions of Yeast Gap1 Permease

PLoS One. 2011 Apr 19;6(4):e18457. doi: 10.1371/journal.pone.0018457.

Abstract

Background: The yeast general amino acid permease Gap1 is a convenient model for studying the intracellular trafficking of membrane proteins. Present at the plasma membrane when the nitrogen source is poor, it undergoes ubiquitin-dependent endocytosis and degradation upon addition of a good nitrogen source, e.g., ammonium. It comprises 12 transmembrane domains (TM) flanked by cytosol-facing N- and C-terminal tails (NT, CT). The NT of Gap1 contains the acceptor lysines for ubiquitylation and its CT includes a sequence essential to exit from the endoplasmic reticulum (ER).

Principal findings: We used alanine-scanning mutagenesis to isolate 64 mutant Gap1 proteins altered in the NT, the CT, or one of the five TM-connecting intracellular loops (L2, -4, -6, -8 and -10). We found 17 mutations (in L2, L8, L10 and CT) impairing Gap1 exit from the ER. Of the 47 mutant proteins reaching the plasma membrane normally, two are unstable and rapidly down-regulated even when the nitrogen source is poor. Six others are totally inactive and another four, altered in a 16-amino-acid sequence in the NT, are resistant to ammonium-induced down-regulation. Finally, a mutation in L6 causes missorting of Gap1 from the secretory pathway to the vacuole. Interestingly, this direct vacuolar sorting seems to be independent of Gap1 ubiquitylation.

Conclusions: This study illustrates the importance of multiple intracellular regions of Gap1 in its secretion, transport activity, and down-regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Amino Acid Sequence
  • Amino Acid Transport Systems / chemistry*
  • Amino Acid Transport Systems / genetics*
  • DNA Mutational Analysis
  • Down-Regulation / drug effects
  • Genes, Fungal / genetics
  • Lysine / metabolism
  • Membrane Transport Proteins / chemistry*
  • Membrane Transport Proteins / genetics*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Phenotype
  • Protein Structure, Tertiary
  • Quaternary Ammonium Compounds / pharmacology
  • Saccharomyces cerevisiae / drug effects
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics*
  • Subcellular Fractions / drug effects
  • Subcellular Fractions / metabolism
  • Ubiquitin / metabolism

Substances

  • Amino Acid Transport Systems
  • GAP1 protein, S cerevisiae
  • Membrane Transport Proteins
  • Mutant Proteins
  • Quaternary Ammonium Compounds
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin
  • Lysine