Tap42-associated protein phosphatase type 2A negatively regulates induction of autophagy.
ABSTRACT Autophagy is a highly conserved degradative process in eukaryotic cells. This process plays an integral role in cellular physiology, and the levels of autophagy must be precisely controlled to prevent cellular dysfunction. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating the induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that Tap42 and protein phosphatase type 2A (PP2A) are involved in the regulation of autophagy in yeast. Temperature-sensitive mutant alleles of TAP42 revealed that autophagy was induced without inactivation of TORC1. Absence of the Tap42-interacting protein Tip41 abolished autophagy induction in the tap42 mutants, whereas overexpression of Tip41 activated autophagy. Furthermore, inactivation of PP2A stimulated autophagy and overexpression of a catalytic subunit of PP2A blocked rapamycin-induced autophagy. Our data support a model in which autophagy is negatively regulated by the Tap42-PP2A pathway.
Article: Mutations in the Saccharomyces cerevisiae type 2A protein phosphatase catalytic subunit reveal roles in cell wall integrity, actin cytoskeleton organization and mitosis.[show abstract] [hide abstract]
ABSTRACT: Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37 degrees), cells dependent solely on pph22 alleles for PP2A function displayed a rapid arrest of proliferation. Ts pph22 mutant cells underwent lysis at 37 degrees, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37 degrees. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24 degrees to 37 degrees, Ts- pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37 degrees, many Ts- pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts- pph22 mutant cells also accumulated aberrant microtubule structures at 37 degrees, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts- pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene.Genetics 03/1997; 145(2):227-41. · 4.01 Impact Factor
Article: Saccharomyces cerevisiae protein phosphatase 2A performs an essential cellular function and is encoded by two genes.[show abstract] [hide abstract]
ABSTRACT: Two genes (PPH21 and PPH22) encoding the yeast homologues of protein serine-threonine phosphatase 2A have been cloned from a Saccharomyces cerevisiae genomic library using a rabbit protein phosphatase 2A cDNA as a hybridization probe. The PPH genes are genetically linked on chromosome IV and are predicted to encode polypeptides each with 74% amino acid sequence identity to rabbit type 2A protein phosphatase, indicating once again the extraordinarily high degree of sequence conservation shown by protein-phosphatases from different species. The two PPH genes show less than 10% amino acid sequence divergence from each other and while disruption of either PPH gene alone is without any major effect, the double disruption is lethal. This indicates that protein phosphatase 2A activity is an essential cellular function in yeast. Measurement of type 2A protein phosphatase activity in yeast strains lacking one or other of the genes indicates that they account for most, if not all, protein phosphatase 2A activity in the cell.The EMBO Journal 01/1991; 9(13):4339-46. · 9.20 Impact Factor
Article: Characteristics of the cholesterol efflux induced by novel seminal phospholipid-binding proteins.[show abstract] [hide abstract]
ABSTRACT: Our recent results indicated that the major proteins of bovine seminal plasma (collectively called BSP proteins) stimulate cholesterol efflux from fibroblasts and that this process shows many differences compared to the efflux induced by apolipoprotein A-I (apoA-I)-containing lipoproteins. The present study was undertaken to investigate the BSP-mediated efflux mechanism. Compared to the slow and constant rate of cholesterol efflux induced by apoA-I-containing lipoproteins, the BSP proteins stimulated a rapid efflux that gradually reached a plateau. The addition of purified BSP proteins after the establishment of the plateau resulted in a further cholesterol efflux indicating that cellular cholesterol was still available for efflux. Incubation of unlabeled fibroblast culture with the spent medium containing BSP-generated lipid ([(3)H]cholesterol) particles obtained after the establishment of the plateau did not result in any cholesterol influx. Therefore, the plateau did not correspond to an equilibrium of the radiolabel between the medium and the cells but rather to a saturation of the efflux particles with cholesterol. Numerous studies have indicated that the cholesterol efflux induced by apoA-I-containing lipoproteins involves cell-surface receptor, caveolae and intracellular cholesterol mobilization. Therefore, we investigated these characteristics for the BSP-mediated cholesterol efflux. Binding of BSP proteins to cells (evaluated by immunoblotting) reached saturation rapidly and remained constant thereafter. However, after several washings the cell-bound BSP proteins were unable to promote significant cholesterol efflux. Both results indicate no correlation of cholesterol efflux with cell binding. Moreover, in comparison to apoA-I-mediated cholesterol efflux, BSP-mediated efflux was not abolished at temperatures below 22 degrees C indicating that the BSP-induced cholesterol efflux does not involve intracellular cholesterol mobilization. High-density lipoprotein- and apoA-I-mediated cholesterol efflux was inhibited by preincubating fibroblasts with progesterone, whereas the cholesterol efflux by BSP proteins was not, indicating that cell-surface caveolae do not participate in BSP-mediated cholesterol efflux. Our results indicate that the mechanism of cholesterol efflux by BSP proteins is unidirectional and is strikingly different from that mediated by apoA-I-containing lipoproteins.Biochimica et Biophysica Acta 09/2000; 1487(1):24-32. · 4.66 Impact Factor