Eric M. Rubenstein

Access Denied: Snf1 Activation Loop Phosphorylation Is Controlled by Availability of the Phosphorylated Threonine 210 to the PP1 Phosphatase

Phosphorylation of the Saccharomyces cerevisiae Snf1 kinase activation loop is determined by the integration of two reaction rates: the rate of phosphorylation by upstream kinases and the rate of dephosphorylation by Glc7. The activities of the Snf1-activating kinases do not appear to be glucose-regulated, since immune complex kinase assays with each of the three Snf1-activating kinases show similar levels of activity when prepared from cells grown in either high or low glucose. In contrast, the dephosphorylation of the Snf1 activation loop was strongly regulated by glucose. When de novo phosphorylation of Snf1 was inhibited, phosphorylation of the Snf1 activation loop was found to be stable in low glucose but rapidly lost upon the addition of glucose. A greater than 10-fold difference in the rates of Snf1 activation loop dephosphorylation was detected. However, the activity of the Glc7-Reg1 phosphatase may not itself be directly regulated by glucose, since the Glc7-Reg1 enzyme was active in low glucose toward another substrate, the transcription factor Mig1. Glucose-mediated regulation of Snf1 activation loop dephosphorylation is controlled by changes in the ability of the Snf1 activation loop to act as a substrate for Glc7.

SUMO-independent in vivo activity of a SUMO-targeted ubiquitin ligase toward a short-lived transcription factor

Many proteins are regulated by ubiquitin-dependent proteolysis. Substrate ubiquitylation can be stimulated by additional post-translational modifications, including small ubiquitin-like modifier (SUMO) conjugation. The recently discovered SUMO-targeted ubiquitin ligases (STUbLs) mediate the latter effect; however, no endogenous substrates of STUbLs that are degraded under normal conditions are known. From a targeted genomic screen, we now identify the yeast STUbL Slx5-Slx8, a heterodimeric RING protein complex, as a key ligase mediating degradation of the MATalpha2 (alpha2) repressor. The ubiquitin-conjugating enzyme Ubc4 was found in the same screen. Surprisingly, mutants with severe defects in SUMO-protein conjugation were not impaired for alpha2 turnover. Unmodified alpha2 also bound to and was ubiquitylated efficiently by Slx5-Slx8. Nevertheless, when we inactivated four SUMO-interacting motifs (SIMs) in Slx5 that together account for its noncovalent SUMO binding, both in vitro Slx5-Slx8-dependent ubiquitylation and in vivo degradation of alpha2 were inhibited. These data identify alpha2 as the first native substrate of the conserved STUbLs, and demonstrate that its STUbL-mediated ubiquitylation does not require SUMO. We suggest that alpha2, and presumably other proteins, have surface features that mimic SUMO, and therefore can directly recruit STUbLs without prior SUMO conjugation.

Snf1 kinase complexes with different beta subunits display stress-dependent preferences for the three Snf1-activating kinases

Three upstream kinases, Pak1, Tos3 and Elm1, are able to activate the Snf1 kinase. Since the Snf1 kinase itself assembles into three complexes that differ in their beta subunit identity, the possibility exists that each upstream kinase might be dedicated to a single isoform of the Snf1 kinase. To test this dedicated activator hypothesis, we generated a series of yeast strains that lacked different combinations of upstream kinases and beta subunits. Cells expressing only one of the three upstream kinases exhibited distinct abilities to activate Snf1, depending on the beta subunit present in the Snf1 kinase complex and the stress imposed on the cells. Pak1 and Gal83 were the most promiscuous. Pak1 was able to activate all three isoforms of the Snf1 kinase under all stress conditions tested. The Gal83 isoform of Snf1 was able to be activated by any of the three upstream kinases under aerobic growth conditions but showed a preference for Pak1 during growth on raffinose. Our results indicate that the three Snf1-activating kinases are not dedicated to specific isoforms of the Snf1 kinase. Instead, the different isoforms of the Snf1 kinase display stress-dependent preferences for the Pak1, Tos3 and Elm1 kinases.

Aberrant substrate engagement of the ER translocon triggers degradation by the Hrd1 ubiquitin ligase

The Hrd1 ubiquitin ligase plays a role in quality control of two substrates associated with the Sec61 translocon.

Mechanisms Regulating the Protein Kinases of Saccharomyces cerevisiae

Reversible protein phosphorylation is a ubiquitous posttranslational modification in all eukaryotes. It is critically involved in the regulation of nearly all cellular processes and signaling pathways. Protein kinases, the enzymes that catalyze the phosphotransfer reaction, constitute one of the

Subunits of the Snf1 Kinase Heterotrimer Show Interdependence for Association and Activity

The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic α-subunit and two non-catalytic subunits, β and γ. The β-subunit is thought to hold the complex together and control subcellular localization whereas the γ-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the α-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1·Snf1 complex requires the β- and γ-subunits in vivo. However, formation of the Sak1·Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the β-subunits do not contain any γ-subunit, indicating that the Snf1 kinase does not form a stable αγ dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified β- and γ-subunits could stimulate the kinase activity of the full-length α-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

N-terminal acetylation of the yeast Derlin Der1 is essential for Hrd1 ubiquitin-ligase activity toward luminal ER substrates.

Up to 70% of yeast proteins are N-terminally acetylated, but in few cases is the function known. The NatB Nα-acetyltransferase is essential for ER-associated degradation of luminal proteins (ERAD-L). Der1, an ERAD-L cofactor of the Hrd1 ubiquitin ligase, is acetylated by NatB and is the only N-acetylation substrate crucial to ERAD-L.

Regulatory Domains of Snf1-Activating Kinases Determine Pathway Specificity

ABSTRACT In Saccharomyces cerevisiae, the Snf1 kinase can be activated by any one of three upstream kinases, Sak1, Tos3, or Elm1. All three Snf1-activating kinases contain serine/threonine kinase domains near their N termini and large C-terminal domains with little sequence conservation and previously unknown function. Deletion of the C-terminal domains of Sak1 and Tos3 greatly reduces their ability to activate the Snf1 pathway. In contrast, deletion of the Elm1 C-terminal domain has no effect on Snf1 signaling but abrogates the ability of Elm1 to participate in the morphogenetic-checkpoint signaling pathway. Thus, the C-terminal domains of Sak1, Tos3, and Elm1 help to determine pathway specificity. Additional deletion mutants of the Sak1 kinase revealed that the N terminus of the protein is essential for Snf1 signaling. The deletion of 43 amino acids from within the N terminus of Sak1 (residues 87 to 129) completely blocks Snf1 signaling and activation loop phosphorylation in vivo. The Sak1 kinase domain (lacking both N-terminal and C-terminal domains) is catalytically active and specific in vitro but is unable to promote Snf1 signaling in vivo when expressed at normal levels. Our studies indicate that the kinase domains of the Snf1-activating kinases are not sufficient by themselves for their proper function and that the nonconserved N-terminal and C-terminal domains are critical for the biological activities of these kinases.

Redundancy and variation in the ubiquitin-mediated proteolytic targeting of a transcription factor

As central components of the intricate networks of eukaryotic gene regulation, transcription factors are frequent targets of ubiquitin-dependent proteolysis. A well-known example is the budding yeast MATa2 (a2) transcriptional repressor, which functions as a master regulator of cell-type determination. Degradation of a2 by the ubiquitin-proteasome system is necessary for a phenotypic switch from one cell type to another. A surprisingly complex set of ubiquitin-protein conjugation mechanisms are involved. One pathway utilizes an integral-membrane ubiquitin ligase (E3) that also functions in endoplasmic reticulum-associated degradation (ERAD). Recently, we showed that a second a2 ubiquitylation pathway uses a heterodimeric E3 that, while able to bind the ubiquitin-like protein SUMO, directly recognizes non-sumoylated a2. Other transcription factors are now also known to be ubiquitylated by multiple mechanisms; as many as a dozen E3s have been implicated in degradation of the human p53 tumor suppressor, for example. We discuss general issues of redundancy and mechanistic variation in protein modification by ubiquitin.

Rkr1/Ltn1 Ubiquitin Ligase-mediated Degradation of Translationally Stalled Endoplasmic Reticulum Proteins

Background: Translationally stalled proteins (including those aberrantly translated beyond their stop codons) pose dangers for eukaryotic cells. Results: The ubiquitin ligase Rkr1/Ltn1 targets translationally stalled ER-associated proteins for degradation. Conclusion: Cytosolic and ER-associated translationally stalled proteins are targeted for destruction by related mechanisms. Significance: Mechanisms regulating the degradation of translationally stalled ER-associated proteins may represent therapeutic targets for human disease. Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane.