A nonconserved Ala401 in the yeast Rsp5 ubiquitin ligase is involved in degradation of Gap1 permease and stress-induced abnormal proteins

Abstract

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.

Fig. 1.

AZC was mainly transported into yeast cells via Gap1. The growth phenotype and AZC content of each strain were examined. Approximately 10⁶ cells of each strain and serial dilutions of 10^–1 to 10^–4 (from left to right) were spotted onto SD+Pro medium or SD+Am plus AZC medium. The plates were incubated at 30°C for 3 days. The intracellular AZC content was measured 2 h after addition of 5 mM AZC in SD+Am liquid medium. The values are means from three independent experiments. Variations in the values are <10%.

Fig. 2.

The recessive mutation of RSP5 enhanced sensitivity to AZC. (A) Various strains were cultivated on SD+Am medium containing 0.1 mM AZC at 30°C for 3 days. (B) Sequence alignment of Rsp5 WW domains. WW1, amino acids 229–266. WW2, amino acids 331–368. WW3, amino acids 387–424. The filled circles at the top indicate the highly conserved residues of the WW domain. The arrow at the bottom shows the mutation found in this study.

Fig. 3.

Gap1 in the rsp5 mutant remained active without ammonium-induced ubiquitination on the plasma membrane. (A) Total RNA (≈50 μg) from strains CKY8 and CHT81 was hybridized to GAP1 and ACT1 probes. Cells were grown on SD+Pro medium before (time = 0) and 60 min after addition of (NH₄)₂SO₄. The GAP1 and ACT1 probes detected the 2.2-kb and the 1.4-kb transcripts, respectively. (B) Immunoblot and permease activity of Gap1 in the membrane-enriched extracts prepared before (time = 0) and at several times after addition of (NH₄)₂SO₄. Gap1 and Put4 activity was measured by incorporation of ¹⁴C-citrulline and ¹⁴C-proline, respectively. To determine the Put4 activity, we used the gap1-disrupted strains derived form CKY8 and CHT81. Each activity before addition of (NH₄)₂SO₄ is given as 100%. (Lower) Levels of the plasma membrane H⁺-ATPase Pma1 as a protein-loading control. (C) Immunoblot of Gap1 from the membrane-enriched extracts prepared before (time = 0) and at several times after addition of (NH₄)₂SO₄. The positions of ubiquitinated forms are indicated with dots.

Fig. 4.

The rsp5 mutants showed hypersensitivity to stresses that induce protein misfolding. The growth phenotypes of various strains were examined. Approximately 10⁶ cells of each strain and serial dilutions of 10^–1 to 10^–4 (from left to right) were spotted and incubated onto SD+Am medium containing the toxic analogue at 30°C for 3 days (A), YPD or SD+Am medium at the indicated temperature for 3 days (B), and the H₂O₂-containing SD medium or SD medium after heat shock treatment (50°C, 6h) at 30°C for 3 days (C).

Fig. 5.

Proposed model for Rsp5-regulated proteolysis. Rsp5 is believed to ubiquitinate Gap1 in an ammonium-induced manner followed by endocytosis and vacuolar degradation (27). Various stress conditions probably lead to accumulate abnormal cellular proteins. The addition of amino acid analogues is believed to cause misfolding of proteins into which it is incorporated. Exposure to H₂O₂, heat shock, and ethanol denatures vulnerable proteins (5, 52). Cell growth by a combination of a rich medium and elevated temperature may disturb the correct folding of newly synthesized proteins. Our results suggest that Rsp5 is involved in degradation of the targeted proteins in these cellular events through ubiquitination. Yeast strains carrying the rsp5 mutation show hypersensitivity to these stresses and defects in spore growth, presumably because of failure of ubiquitination-triggered degradation of these proteins.