Papers by Mark Hochstrasser
Journal of Biological Chemistry, 2016
Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four hetero... more Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (1, 2, and 5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, 5 (Doa3/Pre2) has a 75-residue propeptide, 5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the 5 propeptide nonetheless causes substantial defects in proteasome maturation. Sequences closer to the cleavage site have critical but redundant roles in both assembly and self-cleavage. A conserved histidine two residues upstream of the autocleavage site strongly promotes processing. Surprisingly, although 5pro is functionally linked to the Ump1 assembly factor, trans-expressed 5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed 5pro mutants; the strongest encoded the 7 proteasome subunit. Previous data suggested that 7 and 5pro have overlapping roles in bringing together two half-proteasomes, but the timing of 7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind 7 incorporation into the half-mer. Our results suggest that 7 insertion precedes half-mer dimerization, and the 7 tail and 5 propeptide have unequal roles in half-mer joining.
Molecular Biology of the Cell, Mar 1, 2018
Like many transcription factors, the yeast protein MATalpha2 (α2) undergoes rapid proteolysis via... more Like many transcription factors, the yeast protein MATalpha2 (α2) undergoes rapid proteolysis via the ubiquitin-proteasome system (UPS). At least two ubiquitylation pathways regulate α2 degradation: one pathway utilizes the ubiquitin ligase (E3) Doa10 and the other the heterodimeric E3 Slx5/Slx8. Doa10 is a transmembrane protein of the endoplasmic reticulum/inner nuclear membrane, whereas Slx5/Slx8 localizes to the nucleus and binds DNA nonspecifically. While a single protein can often be ubiquitylated by multiple pathways, the reasons for this "division of labor" are not well understood. Here we show that α2 mutants with impaired DNA binding become inaccessible to the Slx5/Slx8 pathway but are still rapidly degraded through efficient shunting to the Doa10 pathway. These results are consistent with the distinct localization of these E3s. We also characterized a novel class of DNA binding-defective α2 variants whose degradation is strongly impaired. Our genetic data suggest that this is due to a gain-of-function interaction that limits their access to Doa10. Together, these results suggest multiple ubiquitin-ligation mechanisms may have evolved to promote rapid destruction of a transcription factor that resides in distinct cellular subcompartments under different conditions. Moreover, gain-of-function mutations, which also occur with oncogenic forms of human transcription factors such as p53, may derail this fail-safe system.
Journal of Biological Chemistry, Jun 1, 2011
In the endoplasmic reticulum (ER), nascent membrane and secreted proteins that are misfolded are ... more In the endoplasmic reticulum (ER), nascent membrane and secreted proteins that are misfolded are retrotranslocated into the cytosol and degraded by the proteasome. For most ER-associated degradation (ERAD) substrates, ubiquitylation is essential for both their retrotranslocation and degradation. Yeast Doa10 is a polytopic membrane ubiquitin ligase (E3) that along with its cognate ubiquitin-conjugating enzymes (E2s), Ubc7 and the C-terminally membrane-anchored Ubc6, makes a major contribution to ER-associated degradation. Ubc6 is also a substrate of Doa10. One highly conserved Doa10 element, the uncharacterized ϳ130-residue TEB4-Doa10 domain, includes three transmembrane helices (TMs). We find that the first of these, TM5, includes an absolutely conserved ⌽P⌽XXG motif that is required for Doa10 function, as well as highly conserved negatively charged glutamate and aspartate residues. The conservative exchange of the TM5 glutamate to aspartate (doa10-E633D) results in complete stabilization of Ubc6 but has little if any effect on other substrates. Unexpectedly, mutating the glutamate to glutamine (doa10-E633Q) specifically accelerates Ubc6 degradation by ϳ5-fold. Other substrates are weakly stabilized in doa10-E633Q cells, consistent with reduced Ubc6 levels. Notably, catalytically inactive ubc6-C87A is degraded in doa10-E633Q but not wild-type cells, but an active version of Ubc6 is required in trans. Fusion of the Ubc6 TM to a soluble protein yields a protein that is degraded in a doa10-E633Q-dependent manner, whereas fusion of the C-terminal TM from an unrelated protein does not. These results suggest that the TEB4-Doa10 domain regulates Doa10 association with the Ubc6 membrane anchor, thereby controlling the degradation rate of the E2.
Ubiquitin signalling: what's in a chain?
Nature Cell Biology, Jul 1, 2004
When cellular proteins are attached to a Lys 48-linked polyubiquitin chain, the proteasome will u... more When cellular proteins are attached to a Lys 48-linked polyubiquitin chain, the proteasome will usually degrade them. But attaching such a chain to a yeast transcription factor inhibits its activity without degradation, raising questions about how polyubiquitination regulates transcriptional activation and why the protein is spared destruction.
A second degradation signal within the short‐lived transcription factor MATalpha2 (937.1)
The FASEB Journal, Apr 1, 2014
The yeast transcriptional repressor MATalpha2 (alpha2) is a short‐lived protein known to be ubiqu... more The yeast transcriptional repressor MATalpha2 (alpha2) is a short‐lived protein known to be ubiquitylated by at least two distinct pathways, one involving the ubiquitin ligase (E3) Doa10 and the other operating with the E3 Slx5/Slx8. Simultaneous perturbation of both pathways leads to considerable stabilization of alpha2 (approximately 6‐fold); however, deletion of either pathway alone only leads to moderate stabilization of alpha2 (approximately 2‐fold). Although a degradation signal within alpha2, known as Deg1, is well established for Doa10‐dependent degradation, little is known about the determinants within alpha2 that direct its Doa10‐independent degradation. Here we report that alpha2 contains an additional degradation signal, termed Deg2, that is non‐overlapping with Deg1 and leads to ubiquitin‐dependent degradation when fused to normally long‐lived proteins. We also report numerous mutations within Deg2 that result in stabilization of alpha2, including several mutations in the homeodomain that impair the ability of alpha2 to interact with DNA. These data further our understanding of one of the best‐studied substrates of the ubiquitin‐proteasome system and highlight a previously encountered link between the ability of a transcription factor to function and its ubiquitin‐dependent degradation.Grant Funding Source: Supported by the U.S. National Institute of General Medical Sciences
New Proteases in a Ubiquitin Stew
Science, Oct 18, 2002
Proteins destined for degradation in the cell9s garbage disposal unit, the proteasome, must first... more Proteins destined for degradation in the cell9s garbage disposal unit, the proteasome, must first receive a ubiquitin tag. In his Perspective, Hochstrasser discusses new work (Vermaet al., Copeet al.) that reveals the vital link between removal of the ubiquitin tag in the proteasome and degradation of the targeted protein. Proteins are deubiquitinated in the proteasome by a zinc-dependent metalloprotease, and protein degradation cannot take place in the absence of this enzyme.
FEBS Letters, Jul 18, 2000
Ligation of proteins to ubiquitin requires activation of ubiquitin by E1, the ubiquitin-activatin... more Ligation of proteins to ubiquitin requires activation of ubiquitin by E1, the ubiquitin-activating enzyme. Mutant alleles of E1 in mammalian cells have been crucial for dissecting the contribution of the ubiquitin system to cell function. Comparable mutants have been unavailable for Saccharomyces cerevisiae. Here we describe the isolation and characterization of a hypomorphic allele of S. cerevisiae E1. Protein modification by ubiquitin is strongly impaired in the mutant, inhibiting degradation of ubiquitin^proteasome pathway substrates as well as ubiquitin-dependent but proteasome-independent degradation of membrane receptors. This allele will be a useful tool for evaluating the ubiquitin-dependence of cellular processes in yeast, even those in which the proteasome is not involved.
Cell Cycle, Apr 1, 2009
The Slx5/Slx8 protein complex, a heterodimeric SUMOtargeted ubiquitin ligase, plays an important ... more The Slx5/Slx8 protein complex, a heterodimeric SUMOtargeted ubiquitin ligase, plays an important role in genomic integrity. Slx5/Slx8 is believed to interact with sumoylated proteins that reside in the nuclei of budding yeast cells. In this complex, Slx5, owing to at least two SUMO interacting motifs (SIMs), has been proposed to be the targeting subunit of the Slx8 ubiquitin ligase. However, little is known about the exact subnuclear localization and targets of Slx5/Slx8. In this study we show that Slx5, but not Slx8, forms prominent nuclear foci. The formation of these foci depends on SUMO and a SIM in Slx5. Therefore, we investigated the subnuclear localization and potential chromatin association of Slx5. Using co-localization studies in live cells and fixed chromatin, we were able to localize Slx5 to DNA damage induced foci of Rad52 and Rad9, two proteins involved in the cellular response to DNA damage. Subsequent chromatin immunoprecipitation (ChIP) studies revealed that Slx5 is associated with HO endonuclease induced chromosome breaks. Surprisingly, real-time PCR analysis of Slx5 ChIPs revealed that the level of Slx5 at HO breaks in an slx8 deletion background is reduced about 4-fold. These results indicate that the DNA-damage targeting of Slx5/Slx8 depends on formation of the heterodimer and that this occurs at a subset of nuclear foci also containing DNA damage repair and checkpoint factors.
Genes & Development, Apr 13, 2010
Many proteins are regulated by ubiquitin-dependent proteolysis. Substrate ubiquitylation can be s... more 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 MATa2 (a2) 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 a2 turnover. Unmodified a2 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 a2 were inhibited. These data identify a2 as the first native substrate of the conserved STUbLs, and demonstrate that its STUbL-mediated ubiquitylation does not require SUMO. We suggest that a2, and presumably other proteins, have surface features that mimic SUMO, and therefore can directly recruit STUbLs without prior SUMO conjugation.
Nature Structural & Molecular Biology, Feb 17, 2008
Nature, Oct 1, 2006
The ubiquitin system targets many cellular proteins. Doa10 (also known as Ssm4), a yeast transmem... more The ubiquitin system targets many cellular proteins. Doa10 (also known as Ssm4), a yeast transmembrane ubiquitin ligase (E3), resides in the endoplasmic reticulum (ER), but it attaches ubiquitin to soluble proteins that concentrate in the nucleus. A central question is how nuclear substrates gain access to an enzyme in the ER. Here we show that Doa10 reaches the inner nuclear membrane. A subcomplex of nuclear pore subunits is important for this transport. Notably, another ER transmembrane E3, Hrd1 (also known as Der3), cannot localize efficiently to the inner nuclear membrane. Tethering Doa10 at the cell periphery inhibits degradation of soluble nuclear substrates but not cytoplasmic ones. If Doa10 is released from these peripheral sites, localization of Doa10 to the nuclear envelope and degradation of its nuclear substrates are restored in parallel. Thus, localization of Doa10 to the inner nuclear membrane is necessary for nuclear substrate degradation. These data indicate that different membrane ubiquitin ligases are spatially sorted within the ER--nuclear envelope membrane system and that this differential localization contributes to their specificity.
Journal of Biological Chemistry, 2021
Biochemical Functions of Ubiquitin and Ubiquitin-like Protein Conjugation
Wiley-VCH Verlag GmbH & Co. KGaA eBooks, Jan 10, 2008
Journal of Biological Chemistry, Jun 1, 2016
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Papers by Mark Hochstrasser