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1.
Figure 7.

Figure 7. From: Essential Role for the Myotubularin-related Phosphatase Ymr1p and the Synaptojanin-like Phosphatases Sjl2p and Sjl3p in Regulation of Phosphatidylinositol 3-Phosphate in Yeast.

Subcellular localization of targeted GFP-Sac1ΔC chimeras. Wild-type cells were transformed with the indicated construct carried on pRS426, which target Sac1p phosphatase activity to the indicated subcellular compartment. Cells were grown to early log phase in selective media and GFP fluorescence was observed as described in Figure 2.

William R. Parrish, et al. Mol Biol Cell. 2004 August;15(8):3567-3579.
2.
Figure 4.

Figure 4. From: Essential Role for the Myotubularin-related Phosphatase Ymr1p and the Synaptojanin-like Phosphatases Sjl2p and Sjl3p in Regulation of Phosphatidylinositol 3-Phosphate in Yeast.

Ymr1p and Sjl3p are required for proper MVB sorting. (A) GFP-Vps27 is redistributed to the vacuolar surface in the ymr1Δ sjl3Δ mutant. Wild-type and ymr1Δ sjl3Δ cells harboring a 2μURA3-marked GFP-VPS27 plasmid were grown to early log phase in selective media and stained with FM4-64 and processed for microscopy as in Figure 2. (B) GFP-CPS mislocalization exemplifies a defect in MVB sorting in the ymr1Δ sjl3Δ mutant. Wild-type and ymr1Δ sjl3Δ cells harboring a 2μURA3-marked GFP-CPS plasmid were grown to early log phase in selective media and stained with FM4-64 and processed for microscopy as described above.

William R. Parrish, et al. Mol Biol Cell. 2004 August;15(8):3567-3579.
3.
Figure 6.

Figure 6. From: Essential Role for the Myotubularin-related Phosphatase Ymr1p and the Synaptojanin-like Phosphatases Sjl2p and Sjl3p in Regulation of Phosphatidylinositol 3-Phosphate in Yeast.

Sac1p domain activity of Sjl2p is required for viability in ymr1Δ sjl3Δ cells. ymr1Δ sjl2ts sjl3Δ cells were transformed with either an empty vector or the indicated sjl2 mutant allele impaired for either Sac1p domain function (C446S) or 5′ phosphatase activity (D850S) carried on pRS415. Transformants were grown to log phase and 10-fold serial dilutions were spotted onto selective plates at either 26 or at 38°C, and growth was scored after 3 d.

William R. Parrish, et al. Mol Biol Cell. 2004 August;15(8):3567-3579.
4.
Figure 5.

Figure 5. From: Essential Role for the Myotubularin-related Phosphatase Ymr1p and the Synaptojanin-like Phosphatases Sjl2p and Sjl3p in Regulation of Phosphatidylinositol 3-Phosphate in Yeast.

CVT pathway sorting is defective in ymr1Δ sjl3Δ cells. (A) Processing of Ape1p through the CVT pathway, but not through macroautophagy, is severely defective in ymr1Δ sjl3Δ cells. Wild-type and ymr1Δ sjl3Δ cells were grown to early log phase and pulse labeled with [35S]methionine for 15 min and chased with an excess of unlabeled amino acids to the indicated time points. Samples were resolved by SDS-PAGE and visualized by autoradiography. The positions of precursor and mature Ape1p are indicated. (B) Atg24p is mislocalized in ymr1Δ sjl3Δ cells. Wild-type and ymr1Δ sjl3Δ cells carrying a chromosomally integrated Atg24-GFP allele were grown to early log phase in complete media and stained with FM4-64 and processed for microscopy as described in Figure 2.

William R. Parrish, et al. Mol Biol Cell. 2004 August;15(8):3567-3579.
5.
Figure 2.

Figure 2. From: Essential Role for the Myotubularin-related Phosphatase Ymr1p and the Synaptojanin-like Phosphatases Sjl2p and Sjl3p in Regulation of Phosphatidylinositol 3-Phosphate in Yeast.

Ymr1p and Sjl3p coordinately regulate cellular levels and the subcellular distribution of PI(3)P. (A) Quantitative comparison of glycero-phosphoinositols generated by wild-type cells (black bars) or ymr1Δ sjl3Δ mutant cells (gray bars). Cells were grown to early log phase in complete media and then labeled with myo-[2-3H]inositol as in Figure 1. Data represent the mean ± SD of three independent experiments. (B) Ymr1p and Sjl3p control the subcellular distribution of PI(3)P. Steady-state localization of the PI(3)P-specific GFP-FYVEEEA1 chimera in wild-type cells and in ymr1Δ sjl3Δ cells. Cells were maintained in early log phase under appropriate selection for 36–40 h and then harvested into YPD. Cells were stained for 10 min at room temperature with FM4-64, excess dye was washed away and cells were chased for 60 min at room temperature. Cells were then washed with YNB on ice, and aliquots were observed by fluorescence microscopy using a Delta Vision deconvolution system.

William R. Parrish, et al. Mol Biol Cell. 2004 August;15(8):3567-3579.
6.
Figure 3.

Figure 3. From: Essential Role for the Myotubularin-related Phosphatase Ymr1p and the Synaptojanin-like Phosphatases Sjl2p and Sjl3p in Regulation of Phosphatidylinositol 3-Phosphate in Yeast.

Ymr1p and Sjl3p regulate vacuolar protein sorting pathways. (A) ymr1Δ sjl3Δ cells display defects in CPY sorting and CPS maturation. Wild-type and ymr1Δ sjl3Δ cells were analyzed by [35S]methionine pulse-chase labeling followed by immunoprecipitation with antibodies directed against CPY (top) as in Figure 1 or CPS (bottom) as described in MATERIALS AND METHODS. The positions of precursor forms and mature forms are indicated. Results are representative of at least two independent experiments. (B) Vps17-GFP is mislocalized in the ymr1Δ sjl3Δ mutant. Wild-type and ymr1Δ sjl3Δ cells carrying a chromosomally integrated VPS17-GFP allele were grown to early log phase in complete media and stained with FM4-64 and subsequently processed for microscopy as in Figure 2. (C) Vps10-GFP is partially mislocalized in the ymr1Δ sjl3Δ mutant. Wild-type and ymr1Δ sjl3Δ cells carrying a chromosomally integrated VPS10-GFP allele were grown to early log phase in complete media and stained with FM4-64 and processed for microscopy as described above. (D) Vps10p is destabilized in the ymr1Δ sjl3Δ mutant. Cells were pulse labeled for 15 min with [35S]methionine and chased with an excess of unlabeled amino acids for the indicated time. Lysates prepared from these cells were immunoprecipitated with anti-bodies specific to the lumenal domain of Vps10p, and samples were resolved by SDS-PAGE and visualized by autoradiography. Results are representative of at least two independent experiments.

William R. Parrish, et al. Mol Biol Cell. 2004 August;15(8):3567-3579.
7.
Figure 1.

Figure 1. From: Essential Role for the Myotubularin-related Phosphatase Ymr1p and the Synaptojanin-like Phosphatases Sjl2p and Sjl3p in Regulation of Phosphatidylinositol 3-Phosphate in Yeast.

Ymr1p metabolizes a pool of PI(3)P involved in vacuole protein sorting. (A) Schematic representation of the domain architecture of Ymr1p as predicted by the Prodom database. (B) Deletion and overexpression of YMR1 alters PI(3)P levels in vivo. YMR1 deletion cells, wild-type cells (SEY6210), and wild-type cells harboring a 2μYMR1 plasmid were grown under appropriate selection to early log phase and labeled with myo-[2-3H]inositol at 26°C for 60 min. Cellular lipids were recovered, deacylated, and separated by HPLC. Levels of deacylated products corresponding to the indicated phosphoinositides are shown as the % of total PI that was labeled. Data represent the mean ± SD of three independent experiments. (C) Overexpression of YMR1 causes secretion of a pool of CPY; however, YMR1 deletion has no effect. Cells were metabolically labeled at 26°C for 10 min with [35S]methionine and then chased in the presence of excess unlabeled amino acids to the indicated time points. Intracellular pools (I) and secreted pools (E) of CPY were immunoprecipitated, and samples were resolved by SDS-PAGE and visualized by autoradiography. Positions of the endoplasmic reticulum-modified (P1), Golgi-modified (P2), and mature (M) CPY forms are shown.

William R. Parrish, et al. Mol Biol Cell. 2004 August;15(8):3567-3579.
8.
Figure 8.

Figure 8. From: Essential Role for the Myotubularin-related Phosphatase Ymr1p and the Synaptojanin-like Phosphatases Sjl2p and Sjl3p in Regulation of Phosphatidylinositol 3-Phosphate in Yeast.

Sac1p domain activity targeted to PI(3)P rescues lethality of the ymr1Δ sjl2Δ sjl3Δ mutant. (A) ymr1ts sjl2Δ sjl3Δ cells were transformed with an empty vector or the indicated GFP-tagged Sac1p domain chimeras carried on pRS426. A phosphatase-inactive mutant (Sac1ΔC-C392S-FYVEEEA1) was generated by site-directed mutagenesis. Transformants were grown to log phase and 10-fold serial dilutions were spotted as in Figure 6A. (B) ymr1Δ sjl2ts sjl3Δ cells were transformed with either 2 μLEU2-marked GFP-SAC1ΔC-FYVEEEA1 or GFP-SAC1ΔC-2 × PHPLC plasmids and tested for their ability to grow on media containing 5-FOA, which selects against cells harboring the URA3-marked sjl2ts plasmid. The PI(3)P-targeted GFP-SAC1ΔC-FYVEEEA1 plasmid supported viability of the resulting ymr1Δ sjl2Δ sjl3Δ triple mutant cells, whereas the PI(4,5)P2-targeted GFP-SAC1ΔC-2 × PHPLC plasmid did not. (C) GFP-Sac1ΔC-FYVEEEA1 chimera reduces PI(3)P and PI(3,5)P2 in ymr1ts sjl2Δ sjl3Δ cells to levels comparable with YMR1. ymr1ts sjl2Δ sjl3Δ cells carrying an empty vector (black bars), the GFP-Sac1ΔC-FYVEEEA1 chimera (gray bars), or wild-type YMR1 (stippled bars) were grown to early log phase under appropriate selection, preincubated at 38°C for 20 min and labeled with myo-[2-3H]inositol for 60 min at 38°C. Samples were prepared for HPLC as in Figure 1. Levels of deacylated products corresponding to the indicated phosphoinositides are represented as percentage of total labeled PI. Data are representative of at least two independent experiments, and the SD was <15% for each data point.

William R. Parrish, et al. Mol Biol Cell. 2004 August;15(8):3567-3579.

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