A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery

Hitchcock, Amy L.; Auld, Kathryn L.; Gygi, Steven P.; Silver, Pamela A.

doi:10.1073/pnas.2135500100

Cited by 149 publications

(126 citation statements)

References 30 publications

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“…8A and unpublished data). These results are consistent with recently published mass spectrometry data (54,55), indicating that the site of Rvs167 ubiquitination is Lys 481 , the penultimate amino acid in the protein.…”

Section: Fig 5 Sla1 Binds To Rvs167 and Synaptojaninsupporting

confidence: 93%

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

Stamenova¹,

Dunn²,

Adler

et al. 2004

Journal of Biological Chemistry

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Sla1 and Rvs167 are yeast proteins required for receptor internalization and organization of the actin cytoskeleton. Here we provide evidence that Sla1 and Rvs167 are orthologues of the mammalian CIN85 and endophilin proteins, respectively, which are required for ligand-stimulated growth factor receptor internalization. Sla1 is similar in domain structure to CIN85 and binds directly to the endophilin-like Rvs167. Akin to CIN85, Sla1 interacts with synaptojanins and a ubiquitin ligase that regulates endocytosis. This ubiquitin ligase, Rsp5, binds directly to both Sla1 and Rvs167. The interaction between Rsp5 and Rvs167 is mediated through Rsp5 WW domains and PXY motifs in the central Gly-Pro-Ala-rich domain of Rvs167. Rvs167 PXY motifs are required for Rsp5-dependent monoubiquitination of Rvs167 on Lys 481 in the Src homology 3 (SH3) domain. Mutation of Lys 481 3 Arg causes cells to grow slowly on medium containing 1 M NaCl, although this phenotype is not due to the defect in ubiquitination caused by the K481R mutation. We propose that Rsp5 interaction with Sla1-Rvs167 promotes Rvs167 ubiquitination and regulates activity of this protein complex. Rvs167 ubiquitination is not required for general function of Rvs167, but may control specific Rvs167 SH3 domain-protein interactions or negatively regulate SH3 domain activity.

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Section: Fig 5 Sla1 Binds To Rvs167 and Synaptojaninsupporting

confidence: 93%

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

Stamenova¹,

Dunn²,

Adler

et al. 2004

Journal of Biological Chemistry

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“…A number of reports have provided indirect links between the UPS and mitochondria (Fisk and Yaffe, 1999;Sutovsky et al, 1999;Hitchcock et al, 2003;Peng et al, 2003;Thompson et al, 2003;Rinaldi et al, 2004;Altmann and Westermann, 2005;Dürr et al, 2006). The best evidence so far for involvement of the UPS in directly regulating mitochondrial outer membrane proteins is in mammals where a specific E3, MARCHV/ MITOL, is implicated in ubiquitylating two components of Table S1) by treating with CHX.…”

Section: Discussionmentioning

confidence: 99%

Ubiquitin–Proteasome-dependent Degradation of a Mitofusin, a Critical Regulator of Mitochondrial Fusion

Cohen

Leboucher

Livnat-Levanon

et al. 2008

MBoC

152

169

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The mitochondrion is a dynamic membranous network whose morphology is conditioned by the equilibrium between ongoing fusion and fission of mitochondrial membranes. In the budding yeast, Saccharomyces cerevisiae, the transmembrane GTPase Fzo1p controls fusion of mitochondrial outer membranes. Deletion or overexpression of Fzo1p have both been shown to alter the mitochondrial fusion process indicating that maintenance of steady-state levels of Fzo1p are required for efficient mitochondrial fusion. Cellular levels of Fzo1p are regulated through degradation of Fzo1p by the F-box protein Mdm30p. How Mdm30p promotes degradation of Fzo1p is currently unknown. We have now determined that during vegetative growth Mdm30p mediates ubiquitylation of Fzo1p and that degradation of Fzo1p is an ubiquitinproteasome-dependent process. In vivo, Mdm30p associates through its F-box motif with other core components of Skp1-Cullin-F-box (SCF) ubiquitin ligases. We show that the resulting SCF Mdm30p ligase promotes ubiquitylation of Fzo1p at mitochondria and its subsequent degradation by the 26S proteasome. These results provide the first demonstration that a cytosolic ubiquitin ligase targets a critical regulatory molecule at the mitochondrial outer membrane. This study provides a framework for developing an understanding of the function of Mdm30p-mediated Fzo1p degradation in the multistep process of mitochondrial fusion.

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“…Based on the hexahistidine-tagged ubiquitin, ubiquitinated proteins were purified and subsequently identified by mass spectrometry. Two other ubiquitin profiling experiments combined this approach with an initial cell fractionation step to isolate membrane-associated ubiquitination substrates (29) or with isolation of a subset of polyubiquitinated proteins on a polyubiquitin-binding affinity column (30). Immunopurification using antibodies directed against ubiquitin was also used in a large scale approach studying ubiquitination in human cells (31).…”

Section: Figmentioning

confidence: 99%

A Tandem Affinity Tag for Two-step Purification under Fully Denaturing Conditions

Tagwerker

Flick

Cui

et al. 2006

Molecular & Cellular Proteomics

319

181

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Tandem affinity strategies reach exceptional protein purification grades and have considerably improved the outcome of mass spectrometry-based proteomic experiments. However, current tandem affinity tags are incompatible with two-step purification under fully denaturing conditions. Such stringent purification conditions are desirable for mass spectrometric analyses of protein modifications as they result in maximal preservation of posttranslational modifications. Here we describe the histidine-biotin (HB) tag, a new tandem affinity tag for twostep purification under denaturing conditions. The HB tag consists of a hexahistidine tag and a bacterially derived in vivo biotinylation signal peptide that induces efficient biotin attachment to the HB tag in yeast and mammalian cells. HB-tagged proteins can be sequentially purified under fully denaturing conditions, such as 8 M urea, by Ni 2؉ chelate chromatography and binding to streptavidin resins. The stringent separation conditions compatible with the HB tag prevent loss of protein modifications, and the high purification grade achieved by the tandem affinity strategy facilitates mass spectrometric analysis of posttranslational modifications. Ubiquitination is a particularly sensitive protein modification that is rapidly lost during purification under native conditions due to ubiquitin hydrolase activity. The HB tag is ideal to study ubiquitination because the denaturing conditions inhibit hydrolase activity, and the tandem affinity strategy greatly reduces nonspecific background. We tested the HB tag in proteome-wide ubiquitin profiling experiments in yeast and identified a number of known ubiquitinated proteins as well as so far unidentified candidate ubiquitination targets. In addition, the stringent purification conditions compatible with the HB tag allow effective mass spectrometric identification of in vivo cross-linked protein com- Mass spectrometric analysis of proteins has tremendously contributed to our understanding of biological systems. Mapping of covalent protein modifications by mass spectrometric approaches has made it possible to identify and rapidly evaluate the biological significance of modifications. In addition, identification of protein complexes by mass spectrometry has allowed investigators to connect cellular pathways and to describe the dynamics of protein complexes (1, 2). These approaches typically require a high degree of purification of proteins or protein complexes. Importantly to get a genuine picture of the in vivo situation it is essential to avoid any changes in protein modification or protein complex composition that might occur during the purification procedure. Two-step purification strategies have been proven to be very effective in reducing nonspecific background, which is particularly important for the analyses of complex protein samples (3). The first widely and successfully used tandem affinity tag was the TAP 1 tag, which consists of the immunoglobulin-interacting domain of Protein A and a calmodulinbinding peptide (CBP) a...

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A subset of membrane-associated proteins is ubiquitinated in response to mutations in the endoplasmic reticulum degradation machinery

Cited by 149 publications

References 30 publications

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

Ubiquitin–Proteasome-dependent Degradation of a Mitofusin, a Critical Regulator of Mitochondrial Fusion

A Tandem Affinity Tag for Two-step Purification under Fully Denaturing Conditions

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