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 AZChypersensitive 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 NH 4 ؉ 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 nitrogenregulated degradation of Gap1. Furthermore, Ala-401 of Rsp5 was considered to have an important role in the ubiquitination of targeted proteins.A ddition of some amino acid analogues can induce a transient physiological stress response in cells comparable to that of heat shock stress (1-3). Most analogues are transported into cells via amino acid permeases and cause misfolding of the proteins as they compete with naturally occurring amino acids. The accumulation of abnormal proteins, in turn, inhibits cell growth. Recently, Trotter and colleagues (4, 5) found that L-azetidine-2-carboxylic acid (AZC), a toxic four-membered ring analogue of L-proline, arrests proliferation in the G 1 phase of the cell cycle by the same mechanism as temperature up-shift. AZC is an unusual imino acid found only in several plants belonging to the Lilaceae family (6, 7), but can replace L-proline in proteins of bacteria and animal cells (8, 9), presumably in those of yeast cells (5). When AZC was added to cells of yeast Saccharomyces cerevisiae growing in minimal medium, cell viability gradually decreased, causing cell death (M. Nomura and H.T., unpublished work).The accumulation of abnormal or misfolded proteins in cells under stress is a serious problem. To overcome it, the following two strategies can be considered: (i) to degrade the proteins through a ubiquitin-proteasome system or (ii) to refold the proteins by molecular chaperones. We isolated AZC-resistant mutants and strains and elucidated the mechanisms of AZC resistance. Lar...

L-Azetidine-2-carboxylic acid (AZC), a toxic four-membered ring analogue of L-proline, is transported into the cells via proline transporters. It causes misfolding of the proteins into which it is incorporated competitively with L-proline and thereby inhibits the growth of the cells. We recently have discovered, on the chromosome of Saccharomyces cerevisiae ⌺1278b, a novel gene MPR1 required for the resistance of ⌺1278 background strains to toxic AZC. This gene was missing in the particular yeast strain used for the genomic sequence determination. Although the protein sequence was homologous to that of the S. cerevisiae transcriptional regulator, Mpr1p did not affect the expression of genes involved in proline uptake. However, gene expression in Escherichia coli and enzymatic analysis showed that the MPR1 gene encodes a novel AZC acetyltransferase, by which L-proline itself and other L-proline analogues are not acetylated. Mpr1p was considered to be a member of the N-acetyltransferase superfamily based on the results of an Alascan mutagenesis through the highly conserved region involved in binding acetyl-CoA in members of the superfamily. Our findings suggest that Mpr1p detoxifies AZC by acetylating it in the cytoplasm. This enzyme might be utilized as a selective marker in a wide variety of organisms, because the cells expressing the MPR1 gene acquire the AZC-resistant phenotype.Some amino acid analogues are known to induce in cells a transient stress response comparable with that of heat shock stress (1-3). The addition of certain amino acid analogues or mild heat shock causes a rapid increase in the synthesis of heat shock proteins in cells. The elevated levels of synthesis for this set of proteins begins to decrease shortly after restoration of the normal amino acids or normal temperature. Therefore, amino acid analogues have proved to be valuable reagents for studying cellular metabolism and the regulation of synthesis of macromolecules in both prokaryotic and eukaryotic cells (1-3). A toxic four-membered ring analogue of L-proline, L-azetidine-2-carboxylic acid (AZC) 1 , is incorporated into proteins competitively with L-proline and causes the synthesis of abnormal misfolded proteins, thereby inhibiting cell growth in both bacterial and animal cells (4 -7). In bacteria such as Escherichia coli and Serratia marcescens, L-proline biosynthesis from Lglutamate is regulated mainly through feedback inhibition by L-proline of ␥-glutamyl kinase, a key enzyme in the pathway (8,9). Because this enzyme is also feedback-inhibited by L-proline analogues such as AZC, L-proline-overproducing strains have been successfully isolated from L-proline-analogue-resistant mutants, which have a mutation in the proB gene coding for ␥-glutamyl kinase that causes desensitization to feedback inhibition (10, 11).To investigate the cryoprotective effect of L-proline on the freezing stress of yeast, we previously isolated AZC-resistant mutants from an L-proline-nonutilizing strain of Saccharomyces cerevisiae (12). In the process of this...