Quantitative Analysis of Endocytosis with Cytoplasmic pHluorin Chimeras

Prosser, Derek C.; Whitworth, Karen; Wendland, Beverly

doi:10.1111/j.1600-0854.2010.01088.x

Cited by 62 publications

(102 citation statements)

References 26 publications

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“…pHluorin can be targeted to the lumen of yeast vacuoles by fusing it to Mup1 (34). Mup1 is a methionine transporter that localizes to the plasma membrane under low levels of methionine (34). However, excess extracellular methionine triggers the endocytosis of Mup1 followed by sorting into multivesicular bodies and trafficking to the vacuole for degradation (42).…”

Section: Resultsmentioning

confidence: 99%

“…Consequently, by using the Mup1-pHluorin chimera, pHluorin accumulates in the vacuole in the presence of methionine (Ref. 34 and Fig. 3A).…”

Section: Resultsmentioning

confidence: 99%

“…We expected a defect in acidification given that past reports showed that quinacrine does not accumulate in the vacuoles of PtdIns(3,5)P 2 -deficient yeast (27,28). We first employed the super-eclipitic pHluorin, a GFP variant in which fluorescence is potently quenched at low pH (34). pHluorin can be targeted to the lumen of yeast vacuoles by fusing it to Mup1 (34).…”

Section: Resultsmentioning

confidence: 99%

“…cDCFDA was used previously to measure the vacuolar pH in Cryptococcus neoformans by fluorimetry (33). In addition, the super-ecliptic pHluorin, a pH-sensitive GFP variant, can be targeted to the vacuolar lumen to query the pH status of yeast vacuoles (34). We also utilized ratiometric imaging of lysosomes labeled with FITC-dextran to quantitate the lysosomal pH (35).…”

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confidence: 99%

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The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole

Choy

Wattson

et al. 2015

Journal of Biological Chemistry

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Background: Based on qualitative pH probes, loss of Fab1/PIKfyve was thought to impair vacuolar/lysosomal acidification. Results: Using several quantitative assays, vacuoles/lysosomes remained acidic in Fab1/PIKfyve-inhibited cells in a V-ATPasedependent manner. Conclusion: Fab1/PIKfyve is not necessary to maintain the steady-state vacuolar/lysosomal pH. Significance: Contrary to current thought, we propose a model in which Fab1/PIKfyve is dispensable to maintain vacuolar/ lysosomal acidification.

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Section: Resultsmentioning

confidence: 99%

“…Consequently, by using the Mup1-pHluorin chimera, pHluorin accumulates in the vacuole in the presence of methionine (Ref. 34 and Fig. 3A).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole

Choy

Wattson

et al. 2015

Journal of Biological Chemistry

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“…To do this, we used a previously established assay of clathrin-dependent endocytosis based on the clathrin adaptor-mediated internalization of Mup1-GFP, a methionine permease (15,18). In control cells (WT and ⌬4-pENT1), within 30 min of glucose starvation Mup1-GFP cell surface levels decreased, and Mup1-GFP appeared in internal structures ( Fig.…”

Section: Plasma Membrane To Vacuole Traffic Is Required For Cell Survmentioning

confidence: 99%

Glucose Starvation Inhibits Autophagy via Vacuolar Hydrolysis and Induces Plasma Membrane Internalization by Down-regulating Recycling

Lang

Martínez-Márquez

Prosser

et al. 2014

Journal of Biological Chemistry

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107

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Background: During energy starvation, cells utilize intracellular resources for survival; however, the resources used during glucose starvation are unknown. Results: In glucose-starved yeast, vacuolar hydrolysis and endocytosis promote survival whereas autophagy is dispensable. Conclusion: Vacuolar hydrolysis blocks autophagy and provides resources for survival during glucose starvation. Significance: This new survival mechanism could protect cells from starvation in many situations.

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A novel bipartite UNC‐101/AP‐1 μ1 binding signal mediates KVS‐4/Kv2.1 somatodendritic distribution in Caenorhabditis elegans

et al. 2015

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Potassium channels such as Kv2.1 are targeted to specific subcellular compartments to fulfill various functions. However, the mechanisms for their localization are poorly understood. Here, we show that KVS‐4/Kv2.1 somatodendritic localization in Caenorhabditis elegansDA9 neuron requires UNC‐101(AP‐1 μ subunit). We define a bipartite sorting signal within KVS‐4 consisting of a C‐terminal EQMIL and N‐terminal WNIIE motifs. The bipartite signal is sufficient to target nonpolarized transmembrane protein MIG‐13 into DA9 somatodendritic compartments. Furthermore, we found that AP‐1 interacts with the bipartite signal through UNC‐101/AP‐1 μ N‐terminal predicted Longin‐like domain. Our results provide new insight into the mechanisms of Kv2.1 post‐Golgi sorting and targeting.

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Quantitative Analysis of Endocytosis with Cytoplasmic pHluorin Chimeras

Cited by 62 publications

References 26 publications

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole

Glucose Starvation Inhibits Autophagy via Vacuolar Hydrolysis and Induces Plasma Membrane Internalization by Down-regulating Recycling

A novel bipartite UNC‐101/AP‐1 μ1 binding signal mediates KVS‐4/Kv2.1 somatodendritic distribution in Caenorhabditis elegans

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