The yeast phosphotyrosyl phosphatase activator protein, yPtpa1/Rrd1, interacts with Sit4 phosphatase to mediate resistance to 4-nitroquinoline-1-oxide and UVA.

We previously reported the isolation of mutants hypersensitive to the genotoxic agent 4-nitroquinoline-1-oxide, a potent inducer of oxidative stress. One of the mutants was defective in a gene designated yPTPA1, encoding a protein related to the human phosphotyrosyl phosphatase activator hPTPA, which is believed to play a role in activating the serine/threonine phosphatase PP2A. Yeast yptpa1Delta mutants are also sensitive to the UVA component of sunlight known to produce reactive oxygen species, suggesting a role for yPtpa1 in oxidative stress response. We now report the characterization of another 4-nitroquinoline-1-oxide-sensitive mutant, EBY20. We show that this mutant is defective in the SIT4 gene encoding a catalytic subunit of the PP2A phosphatases and that sit4Delta mutants exhibit hypersensitivity to 4-nitroquinoline-1-oxide and UVA, but not to UVC at 254 nm. Like the yptpa1Delta mutants, sit4Delta mutants are also defective in the repair of 4-nitroquinoline-1-oxide-induced DNA lesions. Genetic analysis revealed that both yPtpa1 and Sit4 function in the same pathway to protect cells against the lethal effects of 4-nitroquinoline-1-oxide and UVA. Moreover, we demonstrate that yPtpa1-affinity columns specifically retain Sit4, confirming a previous report that these two proteins indeed belong to a complex. Cellular localization studies using GFP-tagged proteins reveals that yPtpa1 is localized to the cytoplasm and the nucleus, while the Sit4 protein shows an intense staining spot in the cytoplasm and diffused staining in this organelle. We suggest that the yPtpa1-Sit4 complex may participate in a novel mechanism that mediates repair of oxidative DNA damage caused by 4-nitroquinoline-1-oxide and UVA.

Pir1p mediates translocation of the yeast Apn1p endonuclease into the mitochondria to maintain genomic stability.

The mitochondrial genome is continuously subject to attack by reactive oxygen species generated through aerobic metabolism. This leads to the formation of a variety of highly genotoxic DNA lesions, including abasic sites. Yeast Apn1p is localized to the nucleus, where it functions to cleave abasic sites, and apn1 Delta mutants are hypersensitive to agents such as methyl methanesulfonate (MMS) that induce abasic sites. Here we demonstrate for the first time that yeast Apn1p is also localized to the mitochondria. We found that Pir1p, initially isolated as a cell wall constituent of unknown function, interacts with the C-terminal end of Apn1p, which bears a bipartite nuclear localization signal. Further analysis revealed that Pir1p is required to cause Apn1p mitochondrial localization, presumably by competing with the nuclear transport machinery. pir1 Delta mutants displayed a striking (approximately 3-fold) increase of Apn1p in the nucleus, which coincided with drastically reduced levels in the mitochondria. To explore the functional consequences of the Apn1p-Pir1p interaction, we measured the rate of mitochondrial mutations in the wild type and pir1 Delta and apn1 Delta mutants. pir1 Delta and apn1 Delta mutants exposed to MMS exhibited 3.6- and 5.8-fold increases, respectively, in the rate of mitochondrial mutations, underscoring the importance of Apn1p in repair of the mitochondrial genome. We conclude that Pir1p interacts with Apn1p, at the level of either the cytoplasm or nucleus, and facilitates Apn1p transport into the mitochondria to repair damaged DNA.

Oestrogen metabolism in lymphangioleiomyomatosis: catechol-O-methyltransferase pathway is not involved.

BACKGROUND: Lymphangioleiomyomatosis (LAM) is an uncommon lung disease for which no effective method of treatment has been found. The predilection of LAM for premenopausal women has led to the assumption that hormonal factors play an important role in the pathogenesis of this disease. The aim of this study was to determine if women with LAM manifest alterations in the catechol-O-methyltransferase (COMT) pathway which is essential for preventing the generation of oestrogen derived reactive oxygen species (ROS). METHODS: Blood samples were collected from 15 women with LAM and compared with appropriate controls. The distribution of high and low activity alleles of COMT was determined with a PCR based RFLP assay. The enzymatic activity of COMT was measured in each sample and the potential presence of a circulating inhibitor of COMT was determined. Since an alteration in the COMT pathway could increase the oxidative stress, the plasma concentration of malondialdehyde (MDA), a secondary product generated from lipid peroxidation, has been used as an internal marker. RESULTS: The distribution of high and low activity alleles of COMT (named COMT(HH), COMT(LL), and COMT(HL)) was similar in the two groups with proportions of 40%, 7%, and 53%, respectively, in the women with LAM and 38%, 6%, and 56% in the control subjects. The mean (SD) COMT activity was 24.2 (12.3) pmol/min/mg protein in women with LAM and 24.1 (6.3) pmol/min/mg protein in the control group. Incubation of plasma from women in the two groups with a preparation of commercial COMT showed that no detectable COMT inhibitor was present. The plasma concentration of MDA in the women with LAM was also not significantly different from control subjects. CONCLUSIONS: This study shows that there are no significant alterations in the COMT pathway of women with LAM. It is therefore unlikely that alterations in oestrogen mediated cell signalling pathways are mediated by oxidants derived from an excess of catecholoestrogens in LAM.