Desumoylation of the Endoplasmic Reticulum Membrane VAP Family Protein Scs2 by Ulp1 and SUMO Regulation of the Inositol Synthesis Pathway

Abstract

Posttranslational protein modification by the ubiquitin-like SUMO protein is critical to eukaryotic cell regulation, but much remains unknown regarding its operation and substrates. Here we report that specific mutations in the Saccharomyces cerevisiae Ulp1 SUMO protease, including its coiled-coil (CC) domain, lead to the accumulation of distinct sumoylated proteins in vivo. A prominent ϳ50-kDa sumoylated protein accumulates in a Ulp1 CC mutant. The protein was identified as Scs2, an endoplasmic reticulum (ER) membrane protein that regulates phosphatidylinositol synthesis and lipid trafficking. Mutation of lysine 180 of Scs2 abolishes its sumoylation. Notably, impairment of either cellular sumoylation or cellular desumoylation mechanisms inhibits cell growth in the absence of inositol and exacerbates the inositol auxotrophy caused by deletion of SCS2. Mutants lacking the Ulp2 SUMO protease are the most severely affected, and this defect was traced to the mutants' impaired ability to induce transcription of INO1, which encodes the rate-limiting enzyme of inositol biosynthesis. Conversely, inositol starvation induces a striking change in the profiles of total cellular SUMO conjugates. These results provide the first evidence of cross-regulation between the SUMO and inositol pathways, including the sumoylation of an ER membrane protein central to phospholipid synthesis and phosphoinositide signaling. The SUMO (small ubiquitin-related modifier) proteins are a family of protein modifiers conserved from the yeast Saccharomyces cerevisiae to humans. Modification of a protein by SUMO (sumoylation) has a variety of outcomes, including alterations of its localization, stability, or interaction with other molecules (18). Increasingly, SUMO is being identified as a key coordinator of protein interaction networks, and disruption to sumoylation has been linked to a variety of disorders, including many cancers and neurodegenerative diseases (25, 41).The yeast S. cerevisiae expresses a single SUMO protein called Smt3. Smt3 is synthesized as an inactive precursor; the three C-terminal residues are removed to yield mature Smt3, which terminates with a pair of Gly residues. Following precursor cleavage, Smt3 enters an enzymatic cascade involving a series of enzymes designated E1, E2, and E3. The heterodimeric E1-activating enzyme Uba2-Aos1 first forms a high-energy thioester bond with the C terminus of Smt3 (22). Activated Smt3 is then transferred to the active-site cysteine of the E2 enzyme Ubc9 (20), from which it is then conjugated to a lysine side chain in a substrate (21). Smt3-substrate conjugation is usually assisted by one of several E3 protein ligases (21, 50). Additional Smt3 proteins can be appended to previously conjugated Smt3 molecules, generating a polySUMO chain.Sumoylation is reversible, and substrates can be desumoylated by specific proteases, termed ubiquitin-like protein-specific proteases (ULPs) (10). S. cerevisiae has two SUMO proteases, Ulp1 and Ulp2, which cleave Smt3 from distinct sets of substrates (29,31...

SUMO Pathway Modulation of Regulatory Protein Binding at the Ribosomal DNA Locus inSaccharomyces cerevisiae

“…However, ulp2D cells have severe and diverse growth defects, including temperature sensitivity, sporulation defects, hypersensitivity to DNA damage, and hypersensitivity to ER stress, implying multiple substrates linked to growth regulation and stress resistance Hochstrasser 1999, 2000;Strunnikov et al 2001;Kroetz et al 2009;Felberbaum et al 2012). Several recent studies, including the present work, have identified novel Ulp2 substrates (Cremona et al 2012;Srikumar et al 2013b), which will allow more precise analyses of the molecular basis for the defects associated with loss of Ulp2.…”

“…A difficulty in studying aspects of polySUMO function in yeast has been the lack of effective affinity reagents for isolating polySUMO-modified proteins. Available antibodies to yeast SUMO (Smt3) are not effective for immunopurification of conjugates, and epitope tagging Smt3, while useful, leads to a strong reduction in SUMO conjugation efficiency (Wohlschlegel et al 2004;Hannich et al 2005;Felberbaum et al 2012). We show in the present work that a 160-residue fragment of the Slx5 STUbL, which bears four tandem SIMs, has high affinity for polySUMO and yeast HMW-SUMO conjugates.…”

Loss of the SUMO protease Ulp2 triggers a specific multichromosome aneuploidy

“…While many phenotypic defects of ulp2 mutants have been documented, including sensitivity to a variety of stresses, genomic instability, and misregulation of the rDNA locus, much remains unknown regarding the physiological functions of Ulp2 (Li and Hochstrasser 2000;Bylebyl et al 2003;Felberbaum et al 2012). Few protein targets for desumoylation by Ulp2 have been documented.…”

Estradiol potentiates 8-OH-DPAT-induced sumoylation of 5-HT1A receptor: Characterization and subcellular distribution of sumoylated 5-HT1A receptors

“…This hypothesis can explain our observation that acute administration of 5-HT 1A -R agonist, 8-OH-DPAT, significantly increased sumoylated 5-HT 1A -Rs. Several proteins have been reported to be sumoylated in the ER (Felberbaum et al, 2012; Majumdar et al, 2011; Yip et al, 2012). For example, sumoylation of transcription factor X box-binding protein 1(XBP1) in the ER results in accumulation of the protein in the ER, reduction of its activity and increases in ER stress responses (Jiang et al, 2012).…”