Uptake of the ATP-Binding Cassette (ABC) Transporter Ste6 into the Yeast Vacuole Is Blocked in the <i>doa4</i> Mutant

Losko, Sascha; Kopp, Frank; Kranz, Andreas; Kölling, Ralf

doi:10.1091/mbc.12.4.1047

Cited by 69 publications

(78 citation statements)

References 32 publications

(51 reference statements)

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“…Doa4 deubiquitinates ubiquitinated cargo proteins on the endosomal membrane before they are incorporated into the lumenal vesicles of MVBs (Dupre and Haguenauer-Tsapis, 2001;Katzmann et al, 2001;Losko et al, 2001). In doa4 mutant yeast cells, the cytoplasmic free Ub pool is depleted, suggesting that Doa4 retrieves Ub molecules to the cytoplasm before they are cotransported with cargo proteins to and degraded in vacuoles (Swaminathan et al, 1999;Amerik et al, 2000).…”

Section: Role Of Ubpy In Egfr Down-regulationmentioning

confidence: 99%

Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

Mizuno

Iura

Mukai

et al. 2005

MBoC

250

264

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Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This "receptor down-regulation" process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.

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Section: Role Of Ubpy In Egfr Down-regulationmentioning

confidence: 99%

Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

Mizuno

Iura

Mukai

et al. 2005

MBoC

250

264

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“…To construct pRK989, the UBP1 fragment of pRK879 was inserted into the 2 -vector YEplac112 (Gietz and Sugino, 1988). To construct pRK590, a 5.5-kb HindIII/SacI STE6 fragment from pRK182 (Losko et al, 2001) was cloned into YEplac112. YEp112 contains a hemagglutinin (HA)-tagged ubiquitin gene under the control of the CUP1 promoter (Hochstrasser et al, 1991).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

“…Pulse-chase experiments and immunoprecipitation were essentially performed as described previously (Losko et al, 2001).…”

Section: Molecular Biology Of the Cell 1320mentioning

confidence: 99%

“…The cells were washed in 10 mM NaN 3 , resuspended in 110 l of lysis buffer (50 mM HEPES, 0.3 M sorbitol, 10 mM NaN 3 , pH 7.5, protease inhibitor cocktail, and 50 g/ml RNase A) and vortexed for 3 min with 400 mg of glass beads. Pulse-chase experiments and immunoprecipitation were essentially performed as described previously (Losko et al, 2001).…”

Section: In Vivo Labeling Of Cells and Immunoprecipitationmentioning

confidence: 99%

“…Many ubiquitin-dependent processes, such as internalization of cell surface proteins (Hicke, 1999), This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04 -05-0425) on January 5, 2005. targeting of membrane proteins to multivesicular bodies (MVBs) (Katzmann et al, 2001;Losko et al, 2001;Reggiori and Pelham, 2001), retrovirus budding (Patnaik et al, 2000;Schubert et al, 2000;Strack et al, 2000), or ER-associated degradation (ERAD) (Hiller et al, 1996;Wiertz et al, 1996) occur at cellular membranes. To identify potential regulators of ubiquitin-dependent processes at membranes, we looked for membrane-associated Dubs among the 16 yeast members of the Ubp family (Kinner and Kö lling, 2003).…”

Section: Introductionmentioning

confidence: 99%

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The Deubiquitinating Enzyme Ubp1 Affects Sorting of the ATP-binding Cassette-Transporter Ste6 in the Endocytic Pathway

Schmitz

Kinner

Kölling

2005

MBoC

Self Cite

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Deubiquitinating enzymes (Dubs) are potential regulators of ubiquitination-dependent processes. Here, we focus on a member of the yeast ubiquitin-specific processing protease (Ubp) family, the Ubp1 protein. We could show that Ubp1 exists in two forms: a longer membrane-anchored form (mUbp1) and a shorter soluble form (sUbp1) that seem to be independently expressed from the same gene. The membrane-associated mUbp1 variant could be localized to the endoplasmic reticulum (ER) membrane by sucrose density gradient centrifugation and by immunofluorescence microscopy. Overexpression of the soluble Ubp1 variant stabilizes the ATP-binding cassette-transporter Ste6, which is transported to the lysosome-like vacuole for degradation, and whose transport is regulated by ubiquitination. Ste6 stabilization was not the result of a general increase in deubiquitination activity, because overexpression of Ubp1 had no effect on the degradation of the ER-associated degradation substrate carboxypeptidase Y* and most importantly on Ste6 ubiquitination itself. Also, overexpression of another yeast Dub, Ubp3, had no effect on Ste6 turnover. This suggests that the Ubp1 target is a component of the protein transport machinery. On Ubp1 overexpression, Ste6 accumulates at the cell surface, which is consistent with a role of Ubp1 at the internalization step of endocytosis or with enhanced recycling to the cell surface from an internal compartment. INTRODUCTIONMany cellular proteins are modified by the attachment of the 76-amino acid polypeptide ubiquitin (Hochstrasser, 1996;Hershko and Ciechanover, 1998). The main role of ubiquitination is to target proteins for degradation either directly by acting as a degradation signal that is recognized by the 26S proteasome or indirectly by sorting membrane proteins into the lysosomal/vacuolar degradation pathway (Hicke, 1999). Ubiquitination of substrate proteins, which occurs by a cascade of enzymatic reactions, is reversible. Ubiquitin can again be removed from proteins by deubiquitinating enzymes (Dubs). A large number of Dubs have been identified in various organisms that are either cysteine proteases or metalloproteases (Verma et al., 2002; Yao and Cohen, 2002). The more classical cysteine proteases can again be divided into two groups: the ubiquitin C-terminal hydrolases (Uchs) that preferentially cleave ubiquitin from peptides and small adducts (e.g., Yuh1 in yeast) and the extremely divergent family of ubiquitin-specific processing proteases (Ubps) that cleave ubiquitin from protein substrates (Hochstrasser, 1996). Of the 17 cysteine-protease-type Dubs in yeast 16 belong to the Ubp class.The degree of ubiquitination seems to be mainly regulated at the level of ubiquitin attachment. However, evidence is accumulating that deubiquitination also can be important for the regulation of ubiquitination levels. Evidence has been presented that the deubiquitinating enzyme Fat facets (Faf), which is involved in eye development in Drosophila, acts as a substrate-specific regulator of ubiquitination of the...

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AKAP350 Is involved in the development of apical “Canalicular” structures in hepatic cells HepG2

Mattaloni

Kolobova

Favre

et al. 2011

Journal Cellular Physiology

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Hepatocytes are epithelial cells whose apical poles constitute the bile canaliculi. The establishment and maintenance of canalicular poles is a finely regulated process that dictates the efficiency of primary bile secretion. Protein kinase A (PKA) modulates this process at different levels. AKAP350 is an A-kinase anchoring protein that scaffolds protein complexes involved in modulating the dynamic structures of the Golgi apparatus and microtubule cytoskeleton, facilitating microtubule nucleation at this organelle. In this study, we evaluated whether AKAP350 is involved in the development of bile canaliculi-like structures in hepatocyte derived HepG2 cells. We found that AKAP350 recruits PKA to the centrosomes and Golgi apparatus in HepG2 cells. De-localization of AKAP350 from these organelles led to reduced apical cell polarization. A decrease in AKAP350 expression inhibited the formation of canalicular structures and impaired F-actin organization at canalicular poles. Furthermore, loss of AKAP350 expression led to diminished polarized expression of the p-glycoprotein (MDR1/ABCB1) at the apical “canalicular” membrane. AKAP350 knock down effects on canalicular structures formation and actin organization could be mimicked by inhibition of Golgi microtubule nucleation by depletion of CLIP associated proteins (CLASPs). Our data reveal that AKAP350 participates in mechanisms which determine the development of canalicular structures as well as accurate canalicular expression of distinct proteins and actin organization, and provide evidence on the involvement of Golgi microtubule nucleation in hepatocyte apical polarization.

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Uptake of the ATP-Binding Cassette (ABC) Transporter Ste6 into the Yeast Vacuole Is Blocked in the doa4 Mutant

Cited by 69 publications

References 32 publications

Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

Regulation of Epidermal Growth Factor Receptor Down-Regulation by UBPY-mediated Deubiquitination at Endosomes

The Deubiquitinating Enzyme Ubp1 Affects Sorting of the ATP-binding Cassette-Transporter Ste6 in the Endocytic Pathway

AKAP350 Is involved in the development of apical “Canalicular” structures in hepatic cells HepG2

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