VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking
Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chape...
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| Veröffentlicht in: | The Journal of cell biology 2016-08, Vol.214 (4), p.445-458 |
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| creator | Zhou, Mo Wiener, Heidi Su, Wenjuan Zhou, Yong Liot, Caroline Ahearn, Ian Hancock, John F Philips, Mark R |
| description | Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells. |
| doi_str_mv | 10.1083/jcb.201604061 |
| format | Article |
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Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.</description><identifier>ISSN: 0021-9525</identifier><identifier>EISSN: 1540-8140</identifier><identifier>DOI: 10.1083/jcb.201604061</identifier><identifier>PMID: 27502489</identifier><identifier>CODEN: JCLBA3</identifier><language>eng</language><publisher>United States: Rockefeller University Press</publisher><subject>Animals ; Cell Line, Tumor ; Cellular biology ; Cytosol - metabolism ; Dogs ; Gene Silencing ; Green Fluorescent Proteins - metabolism ; GTP Phosphohydrolases - metabolism ; Guanosine Triphosphate - metabolism ; HEK293 Cells ; Humans ; Jurkat Cells ; Lipoylation ; Madin Darby Canine Kidney Cells ; Mass spectrometry ; Melanoma ; Melanoma - metabolism ; Membrane Proteins - metabolism ; Membranes ; Molecular Weight ; Mutant Proteins - metabolism ; Protein Binding ; Protein Prenylation ; Protein Transport ; Proteins ; Subcellular Fractions - metabolism ; Vesicular Transport Proteins - metabolism</subject><ispartof>The Journal of cell biology, 2016-08, Vol.214 (4), p.445-458</ispartof><rights>2016 Zhou et al.</rights><rights>Copyright Rockefeller University Press Aug 15, 2016</rights><rights>2016 Zhou et al. 2016</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c448t-7de9eb437b6545356ec91a6373f6a946e84d10110e5b94ddc401b9e340a078e23</citedby><cites>FETCH-LOGICAL-c448t-7de9eb437b6545356ec91a6373f6a946e84d10110e5b94ddc401b9e340a078e23</cites><orcidid>0000-0002-2275-137X ; 0000-0002-1179-8156 ; 0000-0002-0214-8151 ; 0000-0002-0416-9132</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27502489$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Zhou, Mo</creatorcontrib><creatorcontrib>Wiener, Heidi</creatorcontrib><creatorcontrib>Su, Wenjuan</creatorcontrib><creatorcontrib>Zhou, Yong</creatorcontrib><creatorcontrib>Liot, Caroline</creatorcontrib><creatorcontrib>Ahearn, Ian</creatorcontrib><creatorcontrib>Hancock, John F</creatorcontrib><creatorcontrib>Philips, Mark R</creatorcontrib><title>VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking</title><title>The Journal of cell biology</title><addtitle>J Cell Biol</addtitle><description>Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. 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Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. 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| subjects | Animals Cell Line, Tumor Cellular biology Cytosol - metabolism Dogs Gene Silencing Green Fluorescent Proteins - metabolism GTP Phosphohydrolases - metabolism Guanosine Triphosphate - metabolism HEK293 Cells Humans Jurkat Cells Lipoylation Madin Darby Canine Kidney Cells Mass spectrometry Melanoma Melanoma - metabolism Membrane Proteins - metabolism Membranes Molecular Weight Mutant Proteins - metabolism Protein Binding Protein Prenylation Protein Transport Proteins Subcellular Fractions - metabolism Vesicular Transport Proteins - metabolism |
| title | VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking |
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