Genome-wide bimolecular fluorescence complementation analysis of SUMO interactome in yeast

Sung, Min Kyung; Lim, Gyubum; Yi, Dae-Gwan; Chang, Yeon Ji; Yang, Eun Bin; Lee, KiYoung; Huh, Won-Ki

doi:10.1101/gr.148346.112

Cited by 51 publications

(49 citation statements)

References 44 publications

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“…Consistent with our findings in C. elegans, the HMGS-1 ortholog ERG13 of the sterol-producing yeast S. cerevisiae is suggested to be both sumoylated (33) and ubiquitinated (34). ERG-13 interacts with ubiquitin (UBI4), ubiquitin-specific protease (UBP3), SUMO (SMT3), and a SUMO protease (ULP1) (35) (Fig. 4D), as well as with various proteasomal subunits.…”

Section: )supporting

confidence: 89%

Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging

Sapir

Tsur

Koorman

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin-proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies.HMG-CoA synthase | sterol synthesis | yeast two-hybrid

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Section: )supporting

confidence: 89%

Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging

Sapir

Tsur

Koorman

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Of the set of 49 unique Sho1p interactors, we attempted to confirm 36 (available in a haploid BiFC-tagged collection) by BiFC (Supplementary Table 1). Haploid strains, expressing the putative interactors at their endogenous loci tagged with the N-terminal fragment of YFP (VN) at their C-terminus [31], were mated to haploid strains expressing Sho1p endogenously tagged at either terminus with the C-terminal fragment of YFP (VC) and the diploid strains examined to detect the presence of YFP fluorescence, which is indicative of a physical interaction. Of the 36 putative Sho1p interactors tested by BiFC, 24 (67%) tested positive for an interaction (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae

Lam

Snider

Rehal

et al. 2015

Journal of Molecular Biology

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Sho1p, an integral membrane protein, plays a vital role in the high-osmolarity glycerol (HOG) mitogen-activated protein kinase pathway in the yeast Saccharomyces cerevisiae. Activated under conditions of high osmotic stress, it interacts with other HOG pathway proteins to mediate cell signaling events, ensuring that yeast cells can adapt and remain viable. In an attempt to further understand how the function of Sho1p is regulated through its protein–protein interactions (PPIs), we identified 49 unique Sho1p PPIs through the use of membrane yeast two-hybrid (MYTH), an assay specifically suited to identify PPIs of full-length integral membrane proteins in their native membrane environment. Secondary validation by literature search, or two complementary PPI assays, confirmed 80% of these interactions, resulting in a high-quality Sho1p interactome. This set of putative PPIs included both previously characterized interactors, along with a large subset of interactors that have not been previously identified as binding to Sho1p. The SH3 domain of Sho1p was found to be important for binding to many of these interactors. One particular novel interactor of interest is the glycerol transporter Fps1p, which was shown to require the SH3 domain of Sho1p for binding via its N-terminal soluble regulatory domain. Furthermore, we found that Fps1p is involved in the positive regulation of Sho1p function and plays a role in the phosphorylation of the downstream kinase Hog1p. This study represents the largest membrane interactome analysis of Sho1p to date and complements past studies on the HOG pathway by increasing our understanding of Sho1p regulation.

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“…To facilitate the application of BiFC assays to the genome-wide analysis of PPIs, Huh and colleagues constructed a S. cerevisiae fusion library, in which each endogenous gene was tagged with VN at the C terminus [90] by switching the C-terminal TAP tag of each strain of the TAP fusion library [93] with VN. This VN fusion library contains 5,911 VN-tagged strains and thus covers 95% of all ORFs annotated in the Saccharomyces genome database (as of April 2001; http://www.yeastgenome.org).…”

Section: Large-scale Applications Of Bifcmentioning

confidence: 99%

“…This VN fusion library contains 5,911 VN-tagged strains and thus covers 95% of all ORFs annotated in the Saccharomyces genome database (as of April 2001; http://www.yeastgenome.org). This VN library was then used in a genome-wide search for proteins that interact with small ubiquitin-related modifier (SUMO) proteins in yeast [90]. The attachment of a SUMO molecule to a target protein has been shown to be important for a variety of biological processes [94].…”

Section: Large-scale Applications Of Bifcmentioning

confidence: 99%

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Bimolecular Fluorescence Complementation (BiFC) Analysis: Advances and Recent Applications for Genome-Wide Interaction Studies

Miller

Kim

Huh

et al. 2015

Journal of Molecular Biology

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201

143

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Complex protein networks are involved in nearly all cellular processes. To uncover these vast networks of protein interactions, various high-throughput screening technologies have been developed. Over the last decade, bimolecular fluorescence complementation (BiFC) assay has been widely used to detect protein-protein interactions (PPIs) in living cells. This technique is based on the reconstitution of a fluorescent protein in vivo. Easy quantification of the BiFC signals allows effective cell-based high-throughput screenings for protein-binding partners and drugs that modulate PPIs. Recently, with the development of large screening libraries, BiFC has been effectively applied for genome-wide PPI studies and has uncovered novel protein interactions, providing new insight into protein functions. In this review, we describe the development of reagents and methods used for BiFC-based screens in yeast, plants, and mammalian cells. We also discuss the advantages and drawbacks of these methods and highlight the application of BiFC in large-scale studies.

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Genome-wide bimolecular fluorescence complementation analysis of SUMO interactome in yeast

Cited by 51 publications

References 44 publications

Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging

Controlled sumoylation of the mevalonate pathway enzyme HMGS-1 regulates metabolism during aging

A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae

Bimolecular Fluorescence Complementation (BiFC) Analysis: Advances and Recent Applications for Genome-Wide Interaction Studies

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