The Mouse SKD1, a Homologue of Yeast Vps4p, Is Required for Normal Endosomal Trafficking and Morphology in Mammalian Cells

Yoshimori, Tamotsu; Yamagata, Fumi; Yamamoto, Akira; Mizushima, Noboru; Kabeya, Yukiko; Nara, Atsuki; Miwako, Ishido; Ohashi, Masato; Ohsumi, Mariko; Ohsumi, Yoshinori

doi:10.1091/mbc.11.2.747

Cited by 181 publications

(190 citation statements)

References 41 publications

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“…SKD1 is a mammalian orthologue of yeast Vps4, an AAA-type ATPase that also participates in the endosomal sorting of ubiquitinated membrane proteins . Overexpression of SKD1 E235Q , a dominant-negative SKD1 mutant lacking ATPase activity, causes the accumulation of endo- somal proteins on morphologically aberrant endosomes (Bishop and Woodman, 2000;Yoshimori et al, 2000). In SKD1 E235Q -overexpressing cells, UBPY also localized to the SKD1 E235Q -positive aberrant endosomes ( Figure 5, D-DЉ, arrowheads).…”

Section: Ubpy Functions At Endosomesmentioning

confidence: 95%

“…The construction of expression vectors for human EGFR (Morino et al, 2004), HA-tagged mouse Hrs (Komada et al, 1997), and green fluorescent protein (GFP)-tagged mouse SKD1 E235Q (Yoshimori et al, 2000) was also described previously. The FLAGtagged monoubiquitin expression vector and the c-Cbl expression vector were provided by Dr. T. Suzuki (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan) and Dr. K. Yokote (Chiba University, Chiba, Japan), respectively.…”

Section: Cdna Expression Vectors and Transfectionmentioning

confidence: 99%

“…UBPY colocalized with overexpressed Hrs on the aberrant endosomes ( Figure 5). Second, yeast Vps4 and its mammalian orthologue SKD1 are AAA-type ATPases dysfunction of which results in the accumulation of endocytosed receptors as well as proteins of the endosomal Ub-sorting machinery on aberrant endosomes (Bishop and Woodman, 2000;Yoshimori et al, 2000;Katzmann et al, 2002). UBPY was also accumulated on aberrant endosomes in cells overexpressing SKD1 E235Q , a dominant-negative SKD1 mutant lacking ATPase activity ( Figure 5).…”

Section: Subcellular Site Of Ubpy-mediated Egfr Deubiquitinationmentioning

confidence: 99%

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

Mizuno

Iura

Mukai

et al. 2005

MBoC

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259

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: Ubpy Functions At Endosomesmentioning

confidence: 95%

Section: Cdna Expression Vectors and Transfectionmentioning

confidence: 99%

Section: Subcellular Site Of Ubpy-mediated Egfr Deubiquitinationmentioning

confidence: 99%

See 1 more Smart Citation

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

Mizuno

Iura

Mukai

et al. 2005

MBoC

Self Cite

259

264

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“…For immuno-electron microscopy, the pre-embedding silverenhanced immunogold method was performed as previously described (Yoshimori et al, 2000). HeLa cells were cultured on plastic coverslips (LF; Sumitomo Bakelite).…”

Section: Immuno-electron Microscopymentioning

confidence: 99%

AMSH, an ESCRT-III Associated Enzyme, Deubiquitinates Cargo on MVB/Late Endosomes

Kyuuma

Kikuchi

Kojima

et al. 2006

Cell Struct. Funct.

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and 6 These two authors contributed equally to this work.ABSTRACT. The appropriate sorting of vesicular cargo, including cell-surface proteins, is critical for many cellular functions. Ubiquitinated cargo is targeted to endosomes and digested by lysosomal enzymes. We previously identified AMSH, a deubiquitination enzyme (DUB), to be involved in vesicular transport. Here, we purified an AMSH-binding protein, CHMP3, which is an ESCRT-III subunit. ESCRT-III functions on maturing endosomes, indicating AMSH might also play a role in MVB/late endosomes. Expression of an AMSH mutant lacking CHMP3-binding ability resulted in aberrant endosomes with accumulations of ubiquitinated cargo. Nevertheless, CHMP3-binding capability was not essential for AMSH's in vitro DUB activity or its endosomal localization, suggesting that, in vivo, the deubiquitination of endosomal cargo is CHMP3-dependent. Ubiquitinated cargo also accumulated on endosomes when catalytically inactive AMSH was expressed or AMSH was depleted. These results suggest that both the DUB activity of AMSH and its CHMP3-binding ability are required to clear ubiquitinated cargo from endosomes.

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“…For labeling of lysosome, living cells were incubated with 100 nM LysoTracker Red DND-99 (Molecular Probes, Eugene, OR) for 30 min at 37°C. In the transferrin uptake assay, living cells were incubated with 50 g/ml TexasRed-transferrin (Molecular Probes) for 30 min at 37°C (Meresse et al, 1995;Yoshimori et al, 2000).…”

Section: Fluorescence Microscopymentioning

confidence: 99%

Rabring7, a Novel Rab7 Target Protein with a RING Finger Motif

Mizuno

Kitamura

Sasaki

2003

MBoC

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Rab7, a member of the Rab family small G proteins, has been shown to regulate intracellular vesicle traffic to late endosome/lysosome and lysosome biogenesis, but the exact roles of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins that function in the exocytic/endocytic pathway. We have isolated a new Rab7 target protein, Rabring7 (Rab7-interacting RING finger protein), using a CytoTrap system. It contains an H2 type RING finger motif at the C termini. Rabring7 shows no homology with RILP, which has been reported as another Rab7 target protein. GST pull-down and coimmunoprecipitation assays demonstrate that Rabring7 specifically binds the GTP-bound form of Rab7 at the N-terminal portion. Rabring7 is found mainly in the cytosol and is recruited efficiently to late endosomes/lysosomes by the GTP-bound form of Rab7 in BHK cells. Overexpression of Rabring7 not only affects epidermal growth factor degradation but also causes the perinuclear aggregation of lysosomes, in which the accumulation of the acidotropic probe LysoTracker is remarkably enhanced. These results suggest that Rabring7 plays crucial roles as a Rab7 target protein in vesicle traffic to late endosome/lysosome and lysosome biogenesis

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The Mouse SKD1, a Homologue of Yeast Vps4p, Is Required for Normal Endosomal Trafficking and Morphology in Mammalian Cells

Cited by 181 publications

References 41 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

AMSH, an ESCRT-III Associated Enzyme, Deubiquitinates Cargo on MVB/Late Endosomes

Rabring7, a Novel Rab7 Target Protein with a RING Finger Motif

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