A toxic l-proline analogue, l-azetidine-2-carboxylic acid (AZC), causes misfolding of the proteins into which it is incorporated competitively with l-proline, thereby inhibiting the growth of the cells. AZC enters budding yeast Saccharomyces cerevisiae cells primarily through the general amino acid permease Gap1, not through the proline-specific permease Put4. We isolated an AZC-hypersensitive mutant that cannot grow even at low concentrations of AZC because of the accumulation of intracellular AZC. By screening through a yeast genomic library, the mutant was found to carry an allele of RSP5 encoding an E3 ubiquitin ligase. A single amino acid change replacing Ala (GCA) at position 401 with Glu (GAA) showed that Ala-401 in the third WW domain (a protein interaction module) is not conserved in the domain. The addition of NH4+ to yeast cells growing on l-proline induced rapid ubiquitination, endocytosis, and vacuolar degradation of the plasma membrane protein Gap1. However, immunoblot and permease assays indicated that Gap1 in the rsp5 mutant remained stable and active on the plasma membrane probably with no ubiquitination, leading to AZC accumulation and hypersensitivity. The rsp5 mutants also showed hypersensitivity to various stresses (toxic amino acid analogues, high temperature in a rich medium, and oxidative treatments) and defects in spore growth. These results suggest that Rsp5 is involved in selective degradation of abnormal proteins and specific proteins for spore growth, in addition to nitrogen-regulated degradation of Gap1. Furthermore, Ala-401 of Rsp5 was considered to have an important role in the ubiquitination of targeted proteins.

• PMID: 14500784
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Hoshikawa C, Shichiri M, Nakamori S, Takagi H

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