[48] Yeast endocytosis assays

Dulić, Vjekoslav; Egerton, Mark; Elguindi, I; Raths, Susan; Singer, Birgit; Riezman, Howard

doi:10.1016/0076-6879(91)94051-d

Cited by 194 publications

(245 citation statements)

References 25 publications

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“…␣-Factor Internalization Assays-All ␣-factor internalization assays with strains expressing Ste2p-Ub3xR chimeras were performed as described (32,35). Internalization half-times were calculated based on exponential curve fits performed with Kaleidagraph Software (Synergy Software, Reading, PA).…”

Section: Methodsmentioning

confidence: 99%

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Distinct Functional Surface Regions on Ubiquitin

Sloper-Mould¹,

Jemc²,

Pickart³

et al. 2001

Journal of Biological Chemistry

232

224

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The characterized functions of the highly conserved polypeptide ubiquitin are to target proteins for proteasome degradation or endocytosis. The formation of a polyubiquitin chain of at least four units is required for efficient proteasome binding. By contrast, monoubiquitin serves as a signal for the endocytosis of plasma membrane proteins. We have defined surface residues that are important for ubiquitin's vital functions in Saccharomyces cerevisiae. Surprisingly, alanine scanning mutagenesis showed that only 16 of ubiquitin's 63 surface residues are essential for vegetative growth in yeast. Most of the essential residues localize to two hydrophobic clusters that participate in proteasome recognition and/or endocytosis. The others reside in or near the tail region, which is important for conjugation and deubiquitination. We also demonstrate that the essential residues comprise two distinct functional surfaces: residues surrounding Phe 4 are required for endocytosis, whereas residues surrounding Ile 44 are required for both endocytosis and proteasome degradation.

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Section: Methodsmentioning

confidence: 99%

“…The purification of 35 S-labeled ␣-factor has previously been described (32). Plasmid LHP585 was constructed by subcloning the CUP1-UBI-CYC1 cassette from YEp96 (33) into YEplac195 (34).…”

Section: Methodsmentioning

confidence: 99%

Distinct Functional Surface Regions on Ubiquitin

Sloper-Mould¹,

Jemc²,

Pickart³

et al. 2001

Journal of Biological Chemistry

232

224

View full text Add to dashboard Cite

show abstract

“…Cells were considered as having depolarized actin patches if six or more patches were found within the mother cell. Lucifer yellow uptake assay was performed essentially as described previously (Dulic et al, 1991). Fluorescence microscopy was performed using Nikon E600 fluorescence microscope and Orca-ER charge-coupled device camera (Hamamatsu, Bridgewater, NJ) controlled by Simple PCI software (Compix, Cranberry, PA).…”

Section: Fluorescence Microscopymentioning

confidence: 99%

Probing the Membrane Environment of the TOR Kinases Reveals Functional Interactions between TORC1, Actin, and Membrane Trafficking inSaccharomyces cerevisiae

Aronova

Wedaman

Anderson

et al. 2007

MBoC

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The TOR kinases are regulators of growth in eukaryotic cells that assemble into two distinct protein complexes, TORC1 and TORC2, where TORC1 is inhibited by the antibiotic rapamycin. Present models favor a view wherein TORC1 regulates cell mass accumulation, and TORC2 regulates spatial aspects of growth, including organization of the actin cytoskeleton. Here, we demonstrate that in yeast both TORC1 and TORC2 fractionate with a novel form of detergentresistant membranes that are distinct from detergent-resistant plasma membrane "rafts." Proteomic analysis of these TOR-associated membranes revealed the presence of regulators of endocytosis and the actin cytoskeleton. Genetic analyses revealed a significant number of interactions between these components and TORC1, demonstrating a functional link between TORC1 and actin/endocytosis-related genes. Moreover, we found that inhibition of TORC1 by rapamycin 1) disrupted actin polarization, 2) delayed actin repolarization after glucose starvation, and 3) delayed accumulation of lucifer yellow within the vacuole. By combining our genetic results with database mining, we constructed a map of interactions that led to the identification of additional genetic interactions between TORC1 and components involved in membrane trafficking. Together, these results reveal the broad scope of cellular processes influenced by TORC1, and they underscore the functional overlap between TORC1 and TORC2.

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“…␣-Factor Uptakes-␣-Factor uptakes (continuous presence) were done as described previously (16). The strains were tested at 24°C.…”

Section: Yeastmentioning

confidence: 99%

“…not bear plasmids were grown in complete media YPUAD (2% glucose, 2% peptone, 1% yeast extract, 40 g/ml uracil, 40 g/ml adenine, and 40 g/ml tryptophan, 2% agar for solid media). Unless mentioned otherwise, strains bearing plasmids were selected on SD minimal media (16). Standard recombinant DNA techniques were used (17).…”

mentioning

confidence: 99%

Rvs161p and Rvs167p, the Two Yeast Amphiphysin Homologs, Function Together in Vivo

Lombardi

Riezman

2001

Journal of Biological Chemistry

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Mutations in RVS161 and RVS167, the two yeast amphiphysin homologs, cause very similar growth phenotypes, a depolarized actin cytoskeleton, and a defect in the internalization step of endocytosis. Rvs161p and Rvs167p have been shown to interact in the two-hybrid system, but their localization in the cell may be different thus raising the question whether the interaction is physiologically relevant. Here we demonstrate that the two proteins function together in vivo. We find that the steady state level of Rvs167p is strongly reduced in an rvs161⌬ strain. Similarly, the level of Rvs161p is strongly reduced in an rvs167⌬ strain. We demonstrate that these reduced protein levels at steady state are due to a decreased stability of either Rvs protein in the absence of the other protein. Furthermore, we find that the amount and ratio of Rvs161p and Rvs167p are critical parameters for receptor-mediated endocytosis. In addition, by using the two-hybrid system we show that the interaction of Rvs167p with actin is not abolished in an abp1⌬ strain suggesting that Abp1p is not essential for this interaction.

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[48] Yeast endocytosis assays

Cited by 194 publications

References 25 publications

Distinct Functional Surface Regions on Ubiquitin

Distinct Functional Surface Regions on Ubiquitin

Probing the Membrane Environment of the TOR Kinases Reveals Functional Interactions between TORC1, Actin, and Membrane Trafficking inSaccharomyces cerevisiae

Rvs161p and Rvs167p, the Two Yeast Amphiphysin Homologs, Function Together in Vivo

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