Papers by Mark Hochstrasser
Journal of Cell Biology, Jun 11, 2012
Critical Reviews in Biochemistry and Molecular Biology, Sep 18, 2014
The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and se... more The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and secreted proteins are inserted into or across cell membranes. Its membrane bilayer and luminal compartments provide a favorable environment for the folding and assembly of thousands of newly synthesized proteins. However, protein folding is intrinsically error-prone, and various stress conditions can further increase levels of protein misfolding and damage, particularly in the ER, which can lead to cellular dysfunction and disease. The ubiquitin-proteasome system (UPS) is responsible for the selective destruction of a vast array of protein substrates, either for protein quality control or to allow rapid changes in the levels of specific regulatory proteins. In this review, we will focus on the components and mechanisms of ER-associated protein degradation (ERAD), an important branch of the UPS. ER membranes extend from subcortical regions of the cell to the nuclear envelope, with its continuous outer and inner membranes; the nuclear envelope is a specialized subdomain of the ER. ERAD presents additional challenges to the UPS beyond those faced with soluble substrates of the cytoplasm and nucleus. These include recognition of sugar modifications that occur in the ER, retrotranslocation of proteins across the membrane bilayer, and transfer of substrates from the ER extraction machinery to the proteasome. Here we review characteristics of ERAD substrate degradation signals (degrons), mechanisms underlying substrate recognition and processing by the ERAD machinery, and ideas on the still unresolved problem of how substrate proteins are moved across and extracted from the ER membrane.
PLOS ONE, Oct 4, 2012
Large polytopic membrane proteins often derive from duplication and fusion of genes for smaller p... more Large polytopic membrane proteins often derive from duplication and fusion of genes for smaller proteins. The reverse process, splitting of a membrane protein by gene fission, is rare and has been studied mainly with artificially split proteins. Fragments of a split membrane protein may associate and reconstitute the function of the larger protein. Most examples of naturally split membrane proteins are from bacteria or eukaryotic organelles, and their exact history is usually poorly understood. Here, we describe a nuclear-encoded split membrane protein, split-Doa10, in the yeast Kluyveromyces lactis. In most species, Doa10 is encoded as a single polypeptide with 12-16 transmembrane helices (TMs), but split-KlDoa10 is encoded as two fragments, with the split occurring between TM2 and TM3. The two fragments assemble into an active ubiquitin-protein ligase. The K. lactis DOA10 locus has two ORFs separated by a 508-bp intervening sequence (IVS). A promoter within the IVS drives expression of the C-terminal KlDoa10 fragment. At least four additional Kluyveromyces species contain an IVS in the DOA10 locus, in contrast to even closely related genera, allowing dating of the fission event to the base of the genus. The upstream Kluyveromyces Doa10 fragment with its N-terminal RING-CH and two TMs resembles many metazoan MARCH (Membrane-Associated RING-CH) and related viral RING-CH proteins, suggesting that gene splitting may have contributed to MARCH enzyme diversification. Split-Doa10 is the first unequivocal case of a split membrane protein where fission occurred in a nuclear-encoded gene. Such a split may allow divergent functions for the individual protein segments.
EMBO Reports, Mar 16, 2012
Genetics, Jan 28, 2016
In this report, we identify cellular targets of Ulp2, one of two Saccharomyces cerevisiae small u... more In this report, we identify cellular targets of Ulp2, one of two Saccharomyces cerevisiae small ubiquitin-related modifier (SUMO) proteases, and investigate the function of SUMO modification of these proteins. PolySUMO conjugates from ulp2D and ulp2D slx5D cells were isolated using an engineered affinity reagent containing the four SUMO-interacting motifs (SIMs) of Slx5, a component of the Slx5/Slx8 SUMO-targeted ubiquitin ligase (STUbL). Two proteins identified, Net1 and Tof2, regulate ribosomal DNA (rDNA) silencing and were found to be hypersumoylated in ulp2D, slx5D, and ulp2D slx5D cells. The increase in sumoylation of Net1 and Tof2 in ulp2D, but not ulp1ts cells, indicates that these nucleolar proteins are specific substrates of Ulp2. Based on quantitative chromatin-immunoprecipitation assays, both Net1 and Tof2 lose binding to their rDNA sites in ulp2D cells and both factors largely regain this association in ulp2D slx5D. A parsimonious interpretation of these results is that hypersumoylation of these proteins causes them to be ubiquitylated by Slx5/ Slx8, impairing their association with rDNA. Fob1, a protein that anchors both Net1 and Tof2 to the replication-fork barrier (RFB) in the rDNA repeats, is sumoylated in wild-type cells, and its modification levels increase specifically in ulp2D cells. Fob1 experiences a 50% reduction in rDNA binding in ulp2D cells, which is also rescued by elimination of Slx5. Additionally, overexpression of Sir2, another RFBassociated factor, suppresses the growth defect of ulp2D cells. Our data suggest that regulation of rDNA regulatory proteins by Ulp2 and the Slx5/Slx8 STUbL may be the cause of multiple ulp2D cellular defects.
Nucleic Acids Research, Nov 24, 2020
Histones are substrates of the SUMO (small ubiquitin-like modifier) conjugation pathway. Several ... more Histones are substrates of the SUMO (small ubiquitin-like modifier) conjugation pathway. Several reports suggest histone sumoylation affects transcription negatively, but paradoxically, our genomewide analysis shows the modification concentrated at many active genes. We find that trans-tail regulation of histone-H2B ubiquitylation and H3K4 dimethylation potentiates subsequent histone sumoylation. Consistent with the known control of the Set3 histone deacetylase complex (HDAC) by H3K4 dimethylation, histone sumoylation directly recruits the Set3 complex to both protein-coding and noncoding RNA (ncRNA) genes via a SUMO-interacting motif in the HDAC Cpr1 subunit. The altered gene expression profile caused by reducing histone sumoylation matches well to the profile in cells lacking Set3. Histone H2B sumoylation and the Set3 HDAC coordinately suppress cryptic ncRNA transcription initiation internal to mRNA genes. Our results reveal an elaborate co-transcriptional histone crosstalk pathway involving the consecutive ubiquitylation, methylation, sumoylation and deacetylation of histones, which maintains transcriptional fidelity by suppressing spurious transcription.
The EMBO Journal, Apr 12, 2007
Proteasomes are responsible for most intracellular protein degradation in eukaryotes. The 20S pro... more Proteasomes are responsible for most intracellular protein degradation in eukaryotes. The 20S proteasome comprises a dyad-symmetric stack of four heptameric rings made from 14 distinct subunits. How it assembles is not understood. Most subunits in the central pair of b-subunit rings are synthesized in precursor form. Normally, the b5 (Doa3) propeptide is essential for yeast proteasome biogenesis, but overproduction of b7 (Pre4) bypasses this requirement. Bypass depends on a unique b7 extension, which contacts the opposing b ring. The resulting proteasomes appear normal but assemble inefficiently, facilitating identification of assembly intermediates. Assembly occurs stepwise into precursor dimers, and intermediates contain the Ump1 assembly factor and a novel complex, Pba1-Pba2. b7 incorporation occurs late and is closely linked to the association of two half-proteasomes. We propose that dimerization is normally driven by the b5 propeptide, an intramolecular chaperone, but b7 addition overcomes an Ump1-dependent assembly checkpoint and stabilizes the precursor dimer.
Molecular Biology of the Cell, Oct 1, 2000
The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rap... more The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is required for rapid degradation of ubiquitin-proteasome pathway substrates. Both genetic and biochemical data suggest that Doa4 acts in this pathway by facilitating ubiquitin recycling from ubiquitinated intermediates targeted to the proteasome. Here we describe the isolation of 12 spontaneous extragenic suppressors of the doa4-1 mutation; these involve seven different genes, six of which were cloned. Surprisingly, all of the cloned DID (Doa4-independent degradation) genes encode components of the vacuolar protein-sorting (Vps) pathway. In particular, all are class E Vps factors, which function in the maturation of a late endosome/prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Four of the six Did proteins are structurally related, suggesting an overlap in function. In wild-type and several vps strains, Doa4 -green fluorescent protein displays a cytoplasmic/nuclear distribution. However, in cells lacking the Vps4/Did6 ATPase, a large fraction of Doa4 -green fluorescent protein, like several other Vps factors, concentrates at the late endosome-like class E compartment adjacent to the vacuole. These results suggest an unanticipated connection between protein deubiquitination and endomembrane protein trafficking in which Doa4 acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole.
Cell, Sep 1, 1996
Recently, the Thermoplasma proteasome crystal structure was solved at 3.4 A ˚ . The Department of... more Recently, the Thermoplasma proteasome crystal structure was solved at 3.4 A ˚ . The Department of Biochemistry and Molecular Biology active sites were localized to the  subunits on the inner The University of Chicago surface of a central chamber in the particle. Importantly, Chicago, Illinois 60637 the walls of the cylinder have no large openings, limiting entry of (unfolded) protein substrates to a set of narrow channels leading from the two ends of the particle. The Summary Thermoplasma proteasome is a threonine protease, with the catalytic Thr residue located at the N-terminus of The eukaryotic 20S proteasome is responsible for the the  subunit . When eukaryotic proteasomes are treated with lactacysassembled and how its distinct active sites are formed tin, an irreversible proteasome inhibitor, two sites in one are not understood. Like other proteasome subunits, of the  subunits are rapidly modified, one site being the yeast Doa3 protein is synthesized in precursor the N-terminal Thr residue (Fenteany et al., 1995). If it form. We show that the N-terminal propeptide is reis assumed that the latter modification is what inactiquired for Doa3 incorporation into the proteasome vates the enzyme, it would imply a comparable catalytic and, remarkably, that the propeptide functions in mechanism exists in eukaryotic and Thermoplasma protrans, suggesting it serves a chaperone-like function teasomes. Only three of the seven different eukaryotic in proteasome biogenesis. Propeptide processing is -type subunits in a given 20S particle appear to have not required for proteasome assembly but is needed all the necessary residues required for peptide bond for maturation of a specific subset of active sites. The hydrolysis . likely nucleophile for these sites is provided by the The Thermoplasma protease also differs from the eu-N-terminal threonine of mature Doa3. Additional data karyotic proteasome in that the archaeal enzyme has a indicate that precursor processing is autocatalytic and single kind of active site, the eukaryotic particle at least requires association of the two halves of the proteathree. The archaeal protease cleaves model peptides some particle, thereby preventing formation of proteoonly after hydrophobic residues (a "chymotrypsin-like" lytic sites until the central hydrolytic chamber has been activity); the eukaryotic protease can, in addition, cleave sealed off from the rest of the cell. peptides after basic residues ("trypsin-like" activity) and after acidic residues ("peptidylglutamyl peptide hydrolyzing" [PGPH] activity). It is not known why the eu-
Nature, Mar 1, 2009
Eukaryotic protein modification by ubiquitin-like proteins (Ubls) controls an enormous range of p... more Eukaryotic protein modification by ubiquitin-like proteins (Ubls) controls an enormous range of physiological processes. Ubl attachments principally regulate interactions with other macromolecules, such as proteasome-substrate binding or recruitment of proteins to chromatin. Different Ubl systems use related enzymes to attach specific Ubls to proteins (or other molecules), and most Ubl attachments are transient. Mounting evidence suggests that Ubl-protein modification evolved from prokaryotic sulfurtransferase systems or related enzymes. Surprisingly, proteins similar to Ubl-conjugating and Ubl-deconjugating enzymes appear to have already become widespread by the time of the last universal common ancestor, suggesting that Ubl-protein conjugation is not a eukaryotic invention.
Experimental and Molecular Medicine, Jun 1, 2020
The ubiquitin family member SUMO is a covalent regulator of proteins that functions in response t... more The ubiquitin family member SUMO is a covalent regulator of proteins that functions in response to various stresses, and defects in SUMO-protein conjugation or deconjugation have been implicated in multiple diseases. The loss of the Ulp2 SUMO protease, which reverses SUMO-protein modifications, in the model eukaryote Saccharomyces cerevisiae is severely detrimental to cell fitness and has emerged as a useful model for studying how cells adapt to SUMO system dysfunction. Both short-term and long-term adaptive mechanisms are triggered depending on the length of time cells spend without this SUMO chain-cleaving enzyme. Such short-term adaptations include a highly specific multichromosome aneuploidy and large changes in ribosomal gene transcription. While aneuploid ulp2Δ cells survive, they suffer severe defects in growth and stress resistance. Over many generations, euploidy is restored, transcriptional programs are adjusted, and specific genetic changes that compensate for the loss of the SUMO protease are observed. These long-term adapted cells grow at normal rates with no detectable defects in stress resistance. In this review, we examine the connections between SUMO and cellular adaptive mechanisms more broadly.
Journal of Biological Chemistry, Nov 1, 2013
Background: New regulators of the ubiquitin-proteasome system (UPS) were sought in yeast. Results... more Background: New regulators of the ubiquitin-proteasome system (UPS) were sought in yeast. Results: Cuz1 (Cdc48-associated ubiquitin-like/zinc finger protein-1) interacts with the Cdc48/p97 ATPase and promotes endoplasmic reticulum-associated degradation. Cuz1 is a highly conserved Cdc48 cofactor that also binds proteasomes, especially in cells exposed to arsenite. Significance: This first characterization of the Cuz1 protein family links it to specific Cdc48/p97 complexes. Regulated protein degradation mediated by the ubiquitinproteasome system (UPS) is critical to eukaryotic protein homeostasis. Often vital to degradation of protein substrates is their disassembly, unfolding, or extraction from membranes. These processes are catalyzed by the conserved AAA-ATPase Cdc48 (also known as p97). Here we characterize the Cuz1 protein (Cdc48-associated UBL/zinc finger protein-1), encoded by a previously uncharacterized arsenite-inducible gene in budding yeast. Cuz1, like its human ortholog ZFAND1, has both an AN1type zinc finger (Zf_AN1) and a divergent ubiquitin-like domain (UBL). We show that Cuz1 modulates Cdc48 function in the UPS. The two proteins directly interact, and the Cuz1 UBL, but not Zf_AN1, is necessary for binding to the Cdc48 N-terminal domain. Cuz1 also associates, albeit more weakly, with the proteasome, and the UBL is dispensable for this interaction. Cuz1proteasome interaction is strongly enhanced by exposure of cells to the environmental toxin arsenite, and in a proteasome mutant, loss of Cuz1 enhances arsenite sensitivity. Whereas loss of Cuz1 alone causes only minor UPS degradation defects, its combination with mutations in the Cdc48 Npl4-Ufd1 complex leads to much greater impairment. Cuz1 helps limit the accumulation of ubiquitin conjugates on both the proteasome and Cdc48, suggesting a possible role in the transfer of ubiquitylated substrates from Cdc48 to the proteasome or in their release from these complexes.
Genes, Jul 25, 2020
Many species of arthropods carry maternally inherited bacterial endosymbionts that can influence ... more Many species of arthropods carry maternally inherited bacterial endosymbionts that can influence host sexual reproduction to benefit the bacterium. The most well-known of such reproductive parasites is Wolbachia pipientis. Wolbachia are obligate intracellular α-proteobacteria found in nearly half of all arthropod species. This success has been attributed in part to their ability to manipulate host reproduction to favor infected females. Cytoplasmic incompatibility (CI), a phenomenon wherein Wolbachia infection renders males sterile when they mate with uninfected females, but not infected females (the rescue mating), appears to be the most common. CI provides a reproductive advantage to infected females in the presence of a threshold level of infected males. The molecular mechanisms of CI and other reproductive manipulations, such as male killing, parthenogenesis, and feminization, have remained mysterious for many decades. It had been proposed by Werren more than two decades ago that CI is caused by a Wolbachia-mediated sperm modification and that rescue is achieved by a Wolbachia-encoded rescue factor in the infected egg. In the past few years, new research has highlighted a set of syntenic Wolbachia gene pairs encoding CI-inducing factors (Cifs) as the key players for the induction of CI and its rescue. Within each Cif pair, the protein encoded by the upstream gene is denoted A and the downstream gene B. To date, two types of Cifs have been characterized based on the enzymatic activity identified in the B protein of each protein pair; one type encodes a deubiquitylase (thus named CI-inducing deubiquitylase or cid), and a second type encodes a nuclease (named CI-inducing nuclease or cin). The CidA and CinA proteins bind tightly and specifically to their respective CidB and CinB partners. In transgenic Drosophila melanogaster, the expression of either the Cid or Cin protein pair in the male germline induces CI and the expression of the cognate A protein in females is sufficient for rescue. With the identity of the Wolbachia CI induction and rescue factors now known, research in the field has turned to directed studies on the molecular mechanisms of CI, which we review here.
Molecular Biology of the Cell, Apr 15, 2009
The SUMO protein is covalently attached to many different substrates throughout the cell. This mo... more The SUMO protein is covalently attached to many different substrates throughout the cell. This modification is rapidly reversed by SUMO proteases. The Saccharomyces cerevisiae SUMO protease Ulp2 is a nuclear protein required for chromosome stability and cell cycle restart after checkpoint arrest. Ulp2 is related to the human SENP6 protease, also a nuclear protein. All members of the Ulp2/SENP6 family of SUMO proteases have large but poorly conserved N-terminal domains (NTDs) adjacent to the catalytic domain. Ulp2 also has a long C-terminal domain (CTD). We show that CTD deletion has modest effects on yeast growth, but poly-SUMO conjugates accumulate. In contrast, the NTD is essential for Ulp2 function and is required for nuclear targeting. Two short, widely separated sequences within the NTD confer nuclear localization. Efficient Ulp2 import into the nucleus requires the -importin Kap95, which functions on classical nuclearlocalization signal (NLS)-bearing substrates. Remarkably, replacement of the entire >400-residue NTD by a heterologous NLS results in near-normal Ulp2 function. These data demonstrate that nuclear localization of Ulp2 is crucial in vivo, yet only small segments of the NTD provide the key functional elements, explaining the minimal sequence conservation of the NTDs in the Ulp2/SENP6 family of enzymes.
Methods in molecular biology, 2009
This chapter will discuss various adaptations of the yeast two-hybrid method for analyzing protei... more This chapter will discuss various adaptations of the yeast two-hybrid method for analyzing protein interactions that can be used to identify small ubiquitin-related modifier (SUMO)-interacting proteins and to determine the nature of the SUMO-protein interactions that occur. SUMO binds to a protein in two different ways, covalently and noncovalently. In a covalent interaction an isopeptide bond forms between the glycine residue at the C-terminus of mature SUMO and a lysine side-chain on the substrate protein. Alternatively, SUMO can interact noncovalently with another protein, usually via insertion of a β strand from a substrate SUMO-interacting motif (SIM) into a hydrophobic groove next to the SUMO β2 strand. By mutating either the C-terminal diglycine motif or amino acids within the β2 strand of SUMO, these respective interactions can be abolished. Expression of two-hybrid SUMO constructs with either of these mutations can help distinguish the type of interaction that occurs between SUMO and a given protein. Sumoylation can be verified by independent methods, such as a SUMO mobility shift assay. Finally, the chapter will compare the two-hybrid approach with mass spectrometric analysis as a way to identify SUMO-interacting proteins.
The EMBO Journal, Jan 22, 2004
The 20S proteasome is made up of four stacked heptameric rings, which in eucaryotes assemble from... more The 20S proteasome is made up of four stacked heptameric rings, which in eucaryotes assemble from 14 different but related subunits. The rules governing subunit assembly and placement are not understood. We show that a different kind of proteasome forms in yeast when the Pre9/a3 subunit is deleted. Purified pre9D proteasomes show a two-fold enrichment for the Pre6/a4 subunit, consistent with the presence of an extra copy of Pre6 in each outer ring. Based on disulfide engineering and structure-guided suppressor analyses, Pre6 takes the position normally occupied by Pre9, a substitution that depends on a network of intersubunit salt bridges. When Arabidopsis PAD1/a4 is expressed in yeast, it complements not only pre6D but also pre6D pre9D mutants; therefore, the plant a4 subunit also can occupy multiple positions in a functional yeast proteasome. Importantly, biogenesis of proteasomes is delayed at an early stage in pre9D cells, suggesting an advantage for Pre9 over Pre6 incorporation at the a3 position that facilitates correct assembly.
Genetics, Jun 1, 1998
The 20S proteasome is the proteolytic complex in eukaryotes responsible for degrading short-lived... more The 20S proteasome is the proteolytic complex in eukaryotes responsible for degrading short-lived and abnormal intracellular proteins, especially those targeted by ubiquitin conjugation. The 700-kD complex exists as a hollow cylinder comprising four stacked rings with the catalytic sites located in the lumen. The two outer rings and the two inner rings are composed of seven different ␣ and  polypeptides, respectively, giving an ␣7/7/7/␣7 symmetric organization. Here we describe the molecular organization of the 20S proteasome from the plant Arabidopsis thaliana. From an analysis of a collection of cDNA and genomic clones, we identified a superfamily of 23 genes encoding all 14 of the Arabidopsis proteasome subunits, designated PAA-PAG and PBA-PBG for Proteasome Alpha and Beta subunits A-G, respectively. Four of the subunits likely are encoded by single genes, and the remaining subunits are encoded by families of at least 2 genes. Expression of the ␣ and  subunit genes appears to be coordinately regulated. Three of the nine Arabidopsis proteasome subunit genes tested, PAC1 (␣3), PAE1 (␣5) and PBC2 (3), could functionally replace their yeast orthologs, providing the first evidence for cross-species complementation of 20S subunit genes. Taken together, these results demonstrate that the 20S proteasome is structurally and functionally conserved among eukaryotes and suggest that the subunit arrangement of the Arabidopsis 20S proteasome is similar if not identical to that recently determined for the yeast complex.
The EMBO Journal, Aug 15, 1997
T/proteasome/ubiquitin/Upb14/ is similar in sequence to the human TRE17 proto-oncogene yeast prod... more T/proteasome/ubiquitin/Upb14/ is similar in sequence to the human TRE17 proto-oncogene yeast product and both are active DUBs , functional homology between the two enzymes has not been established. The function of many of the other
Nature Reviews Molecular Cell Biology, Nov 23, 2012
Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins is highly dynamic, and bo... more Covalent attachment of small ubiquitin-like modifier (SUMO) to proteins is highly dynamic, and both SUMO-protein conjugation and cleavage can be regulated. Protein desumoylation is performed by SUMO proteases, which control cellular mechanisms ranging from transcription and cell division to ribosome biogenesis. Recent advances include the discovery of two novel classes of SUMO proteases, insights regarding SUMO protease specificity, and revelations of previously unappreciated SUMO protease functions in several key cellular pathways. These developments, together with new connections between SUMO proteases and the recently discovered SUMOtargeted ubiquitin ligases (STUbLs), make this an exciting period for the study of these enzymes.
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Papers by Mark Hochstrasser