Changes in gene dosage of the YAP) gene, encoding the yAP-1 transcriptional regulatory protein, cause profound alterations in cellular drug and metal resistance. Previous studies on yAP-1 action in yeast cells have used the AP-1 response element (ARE) from simian virus 40 as an artificial site for yAP-i-mediated transcriptional activation. No authentic yeast target sites for control of gene expression by yAP-i are known.Here we show that the GSHI gene, encoding y-glutamylcysteine synthetase, is transcriptionally responsive to the yAP-i protein. GSHI encodes the rate-limiting step in yeast glutathione biosynthesis and contains within its promoter region a DNA element that matches the ARE in 11 of 12 positions. The GSHI yAP-i response element (YRE) was recognized by yAP-i protein in vitro. Northern (RNA) blot analysis showed that GSHI mRNA levels were responsive to YAP) gene dosage. A site-directed mutation in the YRE that blocked yAP-1 binding in vitro prevented the mutant GSHI promoter from responding to elevation in YAP] gene dosage. A Agshl mutant strain was constructed and unable to grow in the absence of exogenous glutathione. A mutant GSHI gene lacking the YRE was unable to confer normal cadmium tolerance, although other yAP-i-mediated phenotypes remained normal. Thus, GSHI is one of several genes that are transcriptionally controlled by yAP-i and influence drug resistance.The Saccharomyces cerevisiae AP-1 protein (yAP-1) was originally identified as a biochemical homolog of mammalian 13). While this biochemical similarity allowed the isolation of the YAP1 gene, the function of the yeast protein was initially difficult to assess. Work from a variety of laboratories has since shown that high-copy-number plasmids carrying the YAP] gene provide dramatic increases in resistance to drugs such as sulfometuron methyl, cycloheximide, 1,10-phenanthroline, 4-nitroquinoline, and cadmium (3,8,16,27,35). Strains lacking a functional YAP1 allele are hypersensitive to oxidative stress (27), 4-nitroquinoline (8), and cadmium (35). Clearly, changes in the copy number of the YAP] gene have pronounced effects on the ability of yeast cells to tolerate drug challenges.While a wide variety of phenotypes that are associated with yAP-1 have been found, none of these phenotypes are thought to be a consequence of the direct action of this factor. The phenotypic influence of yAP-1 on cells is believed to be mediated via this protein acting as a transcriptional regulatory molecule. We have found that mutations in the YAP] gene that inactivate the ability of the gene product to serve as a positive regulator of transcription also block the normal appearance of drug resistance mediated by yAP-1 (34). Thus, the effects of yAP-1 on drug resistance are likely to be mediated by the products of yAP-1-regulated genes. The large number of phenotypes produced by changes in YAP] gene dosage suggests that several different genes are under of yAP-1 control. However, no yAP-1 target gene has yet been identified.In the work described here, we find th...
Integrin-associated protein (IAP, CD47) is a plasma membrane receptor for thrombospondins and signal regulatory proteins (SIRPs) that has an essential role in host defense through its association with integrins. The IAP gene encodes alternatively spliced carboxyterminal cytoplasmic tails that have no previously described function. IAP cytoplasmic tails can bind two related proteins that mediate interaction between IAP and vimentin-containing intermediate filaments, named proteins linking IAP with cytoskeleton (PLICs). Integrins interact with PLICs indirectly, through IAP. Transfection of PLICs induces redistribution of vimentin and cell spreading in IAP-expressing cells. This novel connection between plasma membrane and cytoskeleton is likely to be significant in many adhesion-dependent cell functions.
Changes in gene dosage of the YAP1 gene, encoding the yAP-1 transcriptional regulatory protein, cause profound alterations in cellular drug and metal resistance. Previous studies on yAP-1 action in yeast cells have used the AP-1 response element (ARE) from simian virus 40 as an artificial site for yAP-1-mediated transcriptional activation. No authentic yeast target sites for control of gene expression by yAP-1 are known. Here we show that the GSH1 gene, encoding gamma-glutamylcysteine synthetase, is transcriptionally responsive to the yAP-1 protein. GSH1 encodes the rate-limiting step in yeast glutathione biosynthesis and contains within its promoter region a DNA element that matches the ARE in 11 of 12 positions. The GSH1 yAP-1 response element (YRE) was recognized by yAP-1 protein in vitro. Northern (RNA) blot analysis showed that GSH1 mRNA levels were responsive to YAP1 gene dosage. A site-directed mutation in the YRE that blocked yAP-1 binding in vitro prevented the mutant GSH1 promoter from responding to elevation in YAP1 gene dosage. A delta gsh1 mutant strain was constructed and unable to grow in the absence of exogenous glutathione. A mutant GSH1 gene lacking the YRE was unable to confer normal cadmium tolerance, although other yAP-1-mediated phenotypes remained normal. Thus, GSH1 is one of several genes that are transcriptionally controlled by yAP-1 and influence drug resistance.
The yeast pleiotropic (multiple drug) resistance gene PDR5 encodes a product with homology to a large number of membrane transport proteins including the mammalian multiple drug resistance family. In this study, we identified four genes on chromosome II that affect the steady-state level of PDR5 transcript in addition to a previously identified positive regulator, PDR1. The genes in question are PDR3, PDR4, PDR7 and PDR9. We also analyzed the interaction between PDR5 and YAP1. YAP1 encodes a positive regulator with a leucine zipper motif that causes pleiotropic drug resistance when overproduced. YAP1-mediated pleiotropic drug resistance is not dependent on the presence of PDR5 and must act through other genes.
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