Molecular characterization of a specific p-nitrophenylphosphatase gene, PHO13, and its mapping by chromosome fragmentation in Saccbaromyces cerevisiae

Kaneko, Yoshinobu; Toh‐e, Akio; Banno, Isao; Oshima, Yasuji

doi:10.1007/bf00260867

Cited by 46 publications

(35 citation statements)

References 37 publications

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“…To disrupt the chromosomal PHO13 locus in DKY6281 (SEY6210 pho8⌬::TRP1), the pho13⌬::URA3 plasmid pPH13 (Kaneko et al, 1989) was digested with EcoRI and transformed into DKY6281 to generate strain JGY1. The plasmid pTS15 (pep4⌬::URA3) (Rothman et al, 1986) was digested with EcoRI and transformed into strain JGY3 to disrupt the PEP4 locus and generate the cvt18⌬ pep4⌬ strain JGY7.…”

Section: Strains and Mediamentioning

confidence: 99%

Cvt18/Gsa12 Is Required for Cytoplasm-to-Vacuole Transport, Pexophagy, and Autophagy inSaccharomyces cerevisiaeandPichia pastoris

Guan

Strømhaug

George

et al. 2001

MBoC

196

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Eukaryotic cells have the ability to degrade proteins and organelles by selective and nonselective modes of micro-and macroautophagy. In addition, there exist both constitutive and regulated forms of autophagy. For example, pexophagy is a selective process for the regulated degradation of peroxisomes by autophagy. Our studies have shown that the differing pathways of autophagy have many molecular events in common. In this article, we have identified a new member in the family of autophagy genes. GSA12 in Pichia pastoris and its Saccharomyces cerevisiae counterpart, CVT18, encode a soluble protein with two WD40 domains. We have shown that these proteins are required for pexophagy and autophagy in P. pastoris and the Cvt pathway, autophagy, and pexophagy in S. cerevisiae. In P. pastoris, Gsa12 appears to be required for an early event in pexophagy. That is, the involution of the vacuole or extension of vacuole arms to engulf the peroxisomes does not occur in the gsa12 mutant. Consistent with its role in vacuole engulfment, we have found that this cytosolic protein is also localized to the vacuole surface. Similarly, Cvt18 displays a subcellular localization that distinguishes it from the characterized proteins required for cytoplasm-to-vacuole delivery pathways.

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Section: Strains and Mediamentioning

confidence: 99%

Cvt18/Gsa12 Is Required for Cytoplasm-to-Vacuole Transport, Pexophagy, and Autophagy inSaccharomyces cerevisiaeandPichia pastoris

Guan

Strømhaug

George

et al. 2001

MBoC

196

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“…When cytosolic preparations from strains other than KgI-1A were used, the cytosolic alkaline phosphatase activities (particularly of the PH013 gen¢ product; Kaneko et al, 1989) had to be removed prior to the alkaline phosphatase activity determination. This was accomplished by two sedimentation steps after fusion reactions were completed.…”

Section: In Vitro Analyses With Isolated Vacuolesmentioning

confidence: 99%

“…After solubilization of the vacuolar membrane, alkaline phosphatase activity is quantified spectrophotometrically using pNPP as a chromogenic substrate. To avoid a high background from cytosolic phosphatases, a double deletion mutant in PH08 and PH013 (the latter encoding a cytosolic pNPP-specific phosphatase; Kaneko et al, 1989) Figure L Scheme of the quantitating vacuole-to-vacuole fusion assay. For details see text.…”

Section: Outline Of the In Vitro Fusion-quantification Assaymentioning

confidence: 99%

G-protein ligands inhibit in vitro reactions of vacuole inheritance.

Haas

Conradt

Wickner

1994

The Journal of Cell Biology

168

209

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Abstract. During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J.Cell Biol. 119:1469-1479). We now report a simple and reliable assay to quantify vacuole-to-vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-tovacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2-1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP-hydrolyzing G proteins may play a key role in the in vitro fusion events.

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“…They used strain S288C for the genome project, but we were not able to test this strain for our study because it lacks selective markers for gene disruption and/or chromosome fragmentation. However, we included in our study strains YPH499 (Sikorski and Hieter, 1989), SH962 (Kaneko et al, 1989) and BY4741 (Brachmann et al, 1998), which are genetically intimate to strain S288C; especially, strain BY4741 was constructed by repeated gene disruption of a spore clone derived from S288C (cf. ATTC catalogue).…”

Section: Discussionmentioning

confidence: 99%

Inverted repeat of a large segment unveiled on the right arm of Saccharomyces cerevisiae chromosome II

Ohnishi

Ono

2005

Yeast

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By means of gene disruption analyses, Saccharomyces cerevisiae strain YPH499 was shown to have two, and only two, copies of ATP3 that encodes the γ -subunit of H + -dependent ATP synthase and locates on the right arm of chromosome II. Linkage analyses of the two distinguishably marked copies of ATP3 indicated that the distance between them was about 43 cM. Since YBR030W, an ORF proximal to ATP3 by a distance of 17 kbp, was also found to be duplicated, we marked them with two distinguishable nutritional markers, which were also distinguishable from those used for marking the two copies of ATP3, and achieved four-point linkage analyses; CEN2 marked with an appropriate nutritional marker gene was included as a reference point. And, the following linkage map was deduced: CEN2-[11 cM]-YBR030Wa-[8 cM]-ATP3a-[47 cM]-ATP3b-[55 cM]-YBR030Wb. From this map, we suspected that a segment spanning at least YBR030W-ATP3 would be inversely duplicated on the right arm of chromosome II. We then carried out chromosome fragmentation analyses, using several laboratory strains including YPH499, and obtained data in accord with our speculation for all strains, although the distance between the two copies of ATP3 varied from 48 kbp to 192 kbp among the strains examined.

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Molecular characterization of a specific p-nitrophenylphosphatase gene, PHO13, and its mapping by chromosome fragmentation in Saccbaromyces cerevisiae

Cited by 46 publications

References 37 publications

Cvt18/Gsa12 Is Required for Cytoplasm-to-Vacuole Transport, Pexophagy, and Autophagy inSaccharomyces cerevisiaeandPichia pastoris

Cvt18/Gsa12 Is Required for Cytoplasm-to-Vacuole Transport, Pexophagy, and Autophagy inSaccharomyces cerevisiaeandPichia pastoris

G-protein ligands inhibit in vitro reactions of vacuole inheritance.

Inverted repeat of a large segment unveiled on the right arm of Saccharomyces cerevisiae chromosome II

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