Yeast Rvb1 and Rvb2 Proteins Oligomerize As a Conformationally Variable Dodecamer with Low Frequency

Jeganathan, Ajitha; Leong, Vivian; Zhao, Liang; Huen, Jennifer; Nano, Nardin; Houry, Walid; Ortega, Joaquı́n

doi:10.1016/j.jmb.2015.01.010

Cited by 19 publications

(26 citation statements)

References 36 publications

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“…Rvbs are predominantly hexameric, but a small population of naturally occurring dodecamers has also been reported (Jeganathan et al, 2015). Our observation that Ino80INS binds predominantly to the DII regions of the Rvbs raised the possibility that Ino80INS may activate the Rvbs’ ATPase activity by altering the oligomeric state of Rvb1/Rvb2.…”

Section: Resultsmentioning

confidence: 99%

“…3A and S3D). Previous work with yeast Rvb1/Rvb2 showed that an N-terminal his-tag promotes dodecamerization, providing a means to study the dodecameric state by native-MS (Cheung et al, 2010; Jeganathan et al, 2015). As expected, his-tagged Rvbs ionized as a dodecamer (Figure 3B).…”

Section: Resultsmentioning

confidence: 99%

“…We next asked whether this correlation exists in a context outside of Ino80INS. Similar to the N-terminal his-tag, replacing the DII domain with a flexible linker also increases dodecamerization of Rvbs, but there is conflicting evidence for how these mutants affect the Rvbs’ ATPase activity in the yeast system (Gorynia et al, 2011; Jeganathan et al, 2015; Niewiarowski et al, 2010). When we deleted the DII domains in Rvb1 and Rvb2, we found that this Rvb1ΔDII/Rvb2ΔDII complex has a molecular weight (341 kDa) that is in between what is expected for a dodecamer (474 kDa) and hexamer (237 kDa) by SEC-MALS (Figure 4A and Table S5).…”

Section: Resultsmentioning

confidence: 99%

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Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones

et al. 2017

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Summary The hexameric AAA+ ATPases Rvb1 and Rvb2 (Rvbs) are essential for diverse processes ranging from metabolic signaling to chromatin remodeling but their functions are unknown. While originally thought to act as helicases, recent proposals suggest that Rvbs act as protein assembly chaperones. However, experimental evidence for chaperone-like behavior is lacking. Here, we identify a potent protein activator of the Rvbs, a domain (Ino80INS) in the Ino80 ATPase subunit, of the INO80 chromatin-remodeling complex. Ino80INS stimulates Rvbs’ ATPase activity by 16-fold while concomitantly promoting their dodecamerization. Using mass spectrometry, cryo-EM, and integrative modeling, we find that Ino80INS binds asymmetrically along the dodecamerization interface, resulting in a conformationally flexible dodecamer that collapses into hexamers upon ATP addition. Our results demonstrate the chaperone-like potential of Rvb1/Rvb2 and suggest a model where binding of multiple clients like Ino80 stimulates ATP-driven cycling between hexamers and dodecamers, providing iterative opportunities for correct subunit assembly.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones

et al. 2017

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“…R2TP is the most complicated of the various Hsp90 co-chaperones identified so far, being a multiprotein complex itself, and possessing components (Rvb1 and Rvb2) that like Hsp90 possess inherent ATPase activity on their own (Jeganathan et al., 2015, Nano and Houry, 2013). The role of the ATPase activity of Rvb1 and Rvb2 in the function of R2TP is very poorly understood.…”

Section: Discussionmentioning

confidence: 99%

“…Association of these hetero-hexamers into dodecamers has been observed in several structural studies (Ewens et al., 2016, Gorynia et al., 2011, Jeganathan et al., 2015, Lakomek et al., 2015, Torreira et al., 2008), but the biological relevance of this larger form is unclear. Regardless of the behavior of the Rvb1-Rvb2 complex in isolation, in the yeast R2TP complex we have structurally characterized here, the Rvb1p and Rvb2p proteins are present unambiguously as a single alternating hetero-hexameric ring (Figure 3).…”

Section: Discussionmentioning

confidence: 99%

The Structure of the R2TP Complex Defines a Platform for Recruiting Diverse Client Proteins to the HSP90 Molecular Chaperone System

et al. 2017

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SummaryThe R2TP complex, comprising the Rvb1p-Rvb2p AAA-ATPases, Tah1p, and Pih1p in yeast, is a specialized Hsp90 co-chaperone required for the assembly and maturation of multi-subunit complexes. These include the small nucleolar ribonucleoproteins, RNA polymerase II, and complexes containing phosphatidylinositol-3-kinase-like kinases. The structure and stoichiometry of yeast R2TP and how it couples to Hsp90 are currently unknown. Here, we determine the 3D organization of yeast R2TP using sedimentation velocity analysis and cryo-electron microscopy. The 359-kDa complex comprises one Rvb1p/Rvb2p hetero-hexamer with domains II (DIIs) forming an open basket that accommodates a single copy of Tah1p-Pih1p. Tah1p-Pih1p binding to multiple DII domains regulates Rvb1p/Rvb2p ATPase activity. Using domain dissection and cross-linking mass spectrometry, we identified a unique region of Pih1p that is essential for interaction with Rvb1p/Rvb2p. These data provide a structural basis for understanding how R2TP couples an Hsp90 dimer to a diverse set of client proteins and complexes.

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Reptin regulates insulin‐stimulated Akt phosphorylation in hepatocellular carcinoma via the regulation of SHP‐1/PTPN6

Raymond

Javary

Breig

et al. 2017

Cell Biochemistry & Function

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Hepatocellular carcinoma (HCC) is the main primary cancer of the liver. Many studies have shown that insulin resistance is a risk factor for HCC. We previously discovered the overexpression and oncogenic role of the Reptin/RUVBL2 ATPase in HCC. Here, we found that Reptin silencing enhanced insulin sensitivity in 2 HCC cell lines, as shown by a large potentiation of insulin-induced AKT phosphorylation on Ser473 and Thr308, and of downstream signalling. Reptin silencing did not affect the tyrosine phosphorylation of the insulin receptor nor of IRS1, but it enhanced the tyrosine phosphorylation of the p85 subunit of PI3K. The expression of the SHP-1/PTPN6 phosphatase, which dephosphorylates p85, was reduced after Reptin depletion. Forced expression of SHP-1 restored a normal AKT phosphorylation after insulin treatment in cells where Reptin was silenced, demonstrating that the downregulation of SHP1 is mechanistically linked to increased Akt phosphorylation. In conclusion, we have uncovered a new function for Reptin in regulating insulin signalling in HCC cells via the regulation of SHP-1 expression. We suggest that the regulation of insulin sensitivity by Reptin contributes to its oncogenic action in the liver.

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Yeast Rvb1 and Rvb2 Proteins Oligomerize As a Conformationally Variable Dodecamer with Low Frequency

Cited by 19 publications

References 36 publications

Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones

Regulation of Rvb1/Rvb2 by a Domain within the INO80 Chromatin Remodeling Complex Implicates the Yeast Rvbs as Protein Assembly Chaperones

The Structure of the R2TP Complex Defines a Platform for Recruiting Diverse Client Proteins to the HSP90 Molecular Chaperone System

Reptin regulates insulin‐stimulated Akt phosphorylation in hepatocellular carcinoma via the regulation of SHP‐1/PTPN6

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