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. 2006 Oct 4;25(19):4413-22.
doi: 10.1038/sj.emboj.7601319. Epub 2006 Sep 14.

Ptc1p regulates cortical ER inheritance via Slt2p

Yunrui Du  1 , Lee Walker, Peter Novick, Susan Ferro-Novick
Affiliations

Ptc1p regulates cortical ER inheritance via Slt2p

Yunrui Du et al. EMBO J. .

Abstract

Studies in the yeast Saccharomyces cerevisiae have shown that the inheritance of endoplasmic reticulum (ER), mitochondria, and vacuoles involves the capture of a tubular structure at the bud tip. Ptc1p, a serine/threonine phosphatase, has previously been shown to regulate mitochondrial inheritance by an unknown mechanism. Ptc1p regulates the high osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway and has also been implicated in the cell wall integrity (CWI) MAPK pathway. Here we show that the loss of Ptc1p or the Ptc1p binding protein, Nbp2p, causes a prominent delay in the delivery of ER tubules to the periphery of daughter cells and results in a dramatic increase in the level of phosphorylated Slt2p, the MAPK in the CWI pathway. Either loss of Slt2p or inhibition of the CWI pathway by addition of sorbitol, suppresses the ER inheritance defect in the ptc1Delta and nbp2Delta mutants. Our findings indicate that Ptc1p and Nbp2p regulate ER inheritance through the CWI MAPK pathway by modulating the MAPK, Slt2p.

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Figures

Figure 1
Figure 1
Deletion of the PTC1 gene causes a severe defect in the delivery of ER segregation tubules to the bud cortex. Cells were grown at 25°C in SC medium. Shown are representative wild-type and ptc1Δ cells expressing the ER markers Hmg1p-GFP (A) or Ssh1p-GFP (B). Merged DIC and fluorescence images reveal the localization of ER in the bud. Open arrows, open arrowheads, arrowheads, and arrows point to new, small, medium, and large buds, respectively. The majority of small ptc1Δ buds only contained ER tubules along the mother–bud axis (C). The ER distribution in 250 wild-type buds expressing Hmg1p-GFP (formula image), 253 wild-type buds expressing Ssh1p-GFP (–•–), 264 ptc1Δ buds expressing Hmg1p-GFP (formula image), and 249 ptc1Δ buds expressing Ssh1p-GFP (–○–) was quantitated. The cortical ER tubular network is normal in ptc1Δ mother cells (D). A stack of images of wild-type (SFNY1625) and ptc1Δ (SFNY1624) diploid cells with focal planes 0.1-μm apart were obtained and deconvolved. Images obtained from the center of the cell and from the cell periphery are shown. Arrowheads point to medium buds and arrows point to large buds. Bars, 5 μm.
Figure 2
Figure 2
ER tubules are transported into newly developed ptc1Δ buds, but display a dramatic delay in their delivery to the bud cortex. Time-lapse images of wild-type (SFNY1625) and ptc1Δ (SFNY1624) diploid cells grown in SC medium at 25°C were acquired at 5 min intervals during 120-min time period as described in the Materials and methods. Ten cells were analyzed for each strain and the dynamics of the ER in representative cells are shown. These selected frames from the time series demonstrate that the release of ER tubules from the bud tip is severely delayed in ptc1Δ buds. Bar, 5 μm.
Figure 3
Figure 3
Mutant ptc1Δ cells display a severe delay in the formation of vacuolar and mitochondrial segregation structures. (A) A plasmid expressing the F0ATP synthase mitochondrial targeting sequence fused to RFP was transformed into wild-type (SFNY1255) and the ptc1Δ mutant (SFNY1610). Mitochondria in the transformed cells were visualized by RFP fluorescence. (B) SFNY1255 and SFNY1610 were stained with the vacuole-specific dye FM4-64. After 1 h of labeling, the cells were washed and incubated for 3 h in SC medium. Open arrowheads, arrowheads, and arrows point to small, medium and large buds, respectively. Bars, 5 μm.
Figure 4
Figure 4
The phosphatase activity of Ptc1p is essential for its function in cortical ER inheritance. (A) Expression of PTC1 from a multicopy plasmid suppressed the ER inheritance defects of ptc1Δ (SFNY1610), whereas expression of the metal binding mutation ptc1D58N did not. The fluorescence and fluorescence-DIC merged images of representative cells with small (the first row), medium (the second row), and large buds (the third row) are shown. (B) ER inheritance is normal in cells lacking other PP2C members (Ptc2p, Ptc3p, and Ptc4p). Wild-type and mutant cells from the ResGen library, expressing Hmg1p-GFP, were grown in SC medium at 25°C and analyzed. PCR analysis confirmed the correct gene was disrupted in each of the strains used in this analysis. Bars, 5 μm.
Figure 5
Figure 5
Both PTC1 and NBP2 display genetic interactions with AUX1(SWA2) and SEC3. Tetrads derived from the sporulation of diploid ptc1Δ/PTC1 sec3Δ/SEC3 (A, top panel), ptc1Δ/PTC1 aux1Δ/AUX1 (B), nbp2Δ/NBP2 sec3Δ/SEC3 (C, top panel), nbp2Δ/NBP2 aux1Δ/AUX1 (D), and ptc1Δ/PTC1 nbp2Δ/NBP2 (E) were dissected on YPD (B, D, E) or SC (A, C) plates and incubated at 25°C for 4 (B, D, E) or 6 days (A, C). The colonies enclosed in squares are the double-deletion mutants. The haploid cells (more than six for each genotype) derived from ptc1Δ/PTC1 sec3Δ/SEC3, or nbp2Δ/NBP2 sec3Δ/SEC3 were struck on SC plates and incubated at 25 and 30°C for 3 days. Representative plates are shown in A, C. The colonies on the representative plates are numbered and underlined on the dissection plates.
Figure 6
Figure 6
Deletion of nbp2 leads to a defect in cortical ER inheritance. Mutant cells lacking Nbp2p and expressing Hmg1p-GFP (A) or Ssh1p-GFP (B) were grown at 25°C in SC medium and assayed. The fluorescence and fluorescence-DIC merged images are shown. The open arrowheads, arrowheads, and arrows indicate small, medium, and large buds, respectively. The percentage of nbp2Δ budded cells that displayed cortical ER inheritance defects is comparable to ptc1Δ cells (C). All wild-type, nbp2Δ, and ptc1Δ strains used in this analysis are isogenic. The graph in (C) shows the ER distribution in 250 wild-type buds expressing Hmg1p-GFP (–▴–), 264 ptc1Δ buds expressing Hmg1p-GFP (formula image), 253 nbp2Δ buds expressing Hmg1p-GFP (–▵–), 234 wild-type buds expressing Ssh1p-GFP (formula image), and 260 nbp2Δ buds expressing Ssh1p-GFP (formula image).Bars, 5 μm.
Figure 7
Figure 7
Inactivation of Slt2p, but not Hog1p, suppresses the ER inheritance defects in ptc1 and nbp2 mutant cells. (A) Both Ptc1p and Nbp2p have been implicated in the CWI MAPK pathway (Huang and Symington, 1995; Ohkuni et al, 2003). Protein kinase C (Pkc1p) acts as an upstream activator of this MAPK cascade. Deletion of ptc1 suppresses mutant phenotypes of pkc1–4 cells whereas deletion of either mkk1 or mkk2 suppresses the ts phenotype of nbp2Δ cells. (B) Deletion of slt2, but not hog1, restored ER inheritance in the ptc1Δ mutant. Shown are representative fluorescence and fluorescence-DIC merged images of the ER distribution in ptc1Δ slt2Δ buds and ptc1Δ hog1Δ cells grown to early log phase in SC medium at 25°C. (C) The ER inheritance defects in ptc1Δ and nbp2Δ cells was suppressed by sorbitol. Isogenic wild-type (SFNY1255), ptc1Δ (SFNY1610), and nbp2Δ (SFNY1685) strains were grown to early log phase and divided into two aliquots. Half the cells were grown at 25°C for 3 h in SC medium in the absence (−sorbitol) or presence of 1 M sorbitol (+sorbitol). Representative fluorescence and fluorescence-DIC merged images of cells with small (open arrow heads), medium (arrow heads), and large buds (arrows) are shown. The open arrow in the first row indicates a new wild-type bud. Quantitation revealed that the deletion of slt2 (D) and sorbitol treatment (E) leads to significant suppression. Key: 117 ptc1Δ slt2Δ buds (formula image), 139 ptc1Δ hog1Δ buds (–○–), 278 wild-type buds cultured in SC+1 M sorbitol (–⧫–), 275 ptc1Δ buds grown in SC medium (formula image), 284 ptc1Δ buds grown in SC+1 M sorbitol (formula image), 296 nbp2Δ buds grown in SC medium (–▵–), 284 nbp2Δ buds grown in SC+1 M sorbitol (–▴–) were analyzed. (F) Deletion of slt2 had no effect on vacuole distribution in ptc1Δ mutant and wild-type cells. The vacuole inheritance defect was analyzed as in Figure 3B. Bars, 5 μm.
Figure 8
Figure 8
Deletion of ptc1 and nbp2 leads to an increase in the level of phosphorylated Slt2p in vivo (A). Slt2p phosphorylation and expression was monitored as described in the Materials and methods. Bos1p (27 kDa) was used as a loading control. Expression of PTC1 from a multicopy plasmid decreased the level of phosphorylated Slt2p in ptc1Δ, whereas expression of the metal binding mutation ptc1D58N did not (B).
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References

    1. Acharya U, Mallabiabarrena A, Acharya JK, Malhotra V (1998) Signaling via mitogen-activated protein kinase kinase (MEK1) is required for Golgi fragmentation during mitosis. Cell 92: 183–192 - PubMed
    1. Andrews PD, Stark MJ (2000) Dynamic, Rho1p-dependent localization of Pkc1p to sites of polarized growth. J Cell Sci 113: 2685–2693 - PubMed
    1. Blumer KJ, Johnson GL, Lange-Carter CA (1994) Mammalian mitogen-activated protein kinase kinase kinase (MEKK) can function in a yeast mitogen-activated protein kinase pathway downstream of protein kinase C. Proc Natl Acad Sci USA 91: 4925–4929 - PMC - PubMed
    1. Boldogh IR, Fehrenbacher KL, Yang HC, Pon LA (2005) Mitochondrial movement and inheritance in budding yeast. Gene 354: 28–36 - PubMed
    1. Bonangelino CJ, Chavez EM, Bonifacino JS (2002) Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. Mol Biol Cell 13: 2486–2501 - PMC - PubMed

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