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Mark Hochstrasser

Publications and source records attributed to Mark Hochstrasser.

At least 19 recordsLinked to original sources
PubMed2006

A conserved late endosome-targeting signal required for Doa4 deubiquitylating enzyme function.

Enzyme specificity in vivo is often controlled by subcellular localization. Yeast Doa4, a deubiquitylating enzyme (DUB), removes ubiquitin from membrane proteins destined for vacuolar degradation. Doa4 is recruited to the late endosome after ESCRT-III (endosomal sorting complex required for transport III) has assembled there. We show that an N-terminal segment of Doa4 is sufficient for endosome association. This domain bears four conserved elements (boxes A-D). Deletion of the most conserved of these, A or B, prevents Doa4 endosomal localization. These mutants cannot sustain ubiquitin-dependent proteolysis even though neither motif is essential for deubiquitylating activity. Ubiquitin-specific processing protease 5 (Ubp5), the closest paralogue of Doa4, has no functional overlap. Ubp5 concentrates at the bud neck; its N-terminal domain is critical for this. Importantly, substitution of the Ubp5 N-terminal domain with that of Doa4 relocalizes the Ubp5 enzyme to endosomes and provides Doa4 function. This is the first demonstration of a physiologically important DUB subcellular localization signal and provides a striking example of the functional diversification of DUB paralogues by the evolution of alternative spatial signals.

Amino Acid Sequence↗
PubMed2006

Spatially regulated ubiquitin ligation by an ER/nuclear membrane ligase.

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.

Endoplasmic Reticulum↗
PubMed2006

An amphipathic helix targets serum and glucocorticoid-induced kinase 1 to the endoplasmic reticulum-associated ubiquitin-conjugation machinery.

Serum- and glucocorticoid-induced kinase 1 (Sgk1) regulates many ion channels and transporters in epithelial cells and promotes cell survival under stress conditions. In this study we demonstrate that Sgk1 is a short-lived protein regulated by the endoplasmic reticulum (ER)-associated degradation system and subcellular localization to the ER. We identified a hydrophobic motif (residues 18-30) as the signal for ER localization and rapid degradation by the ubiquitin (Ub)/proteasome pathway in both yeast and mammalian cells. Deletion or reduction of hydrophobicity of the motif redistributes Sgk1 to the cytosol and nucleus and markedly increases its half-life. We determined that the Ub-conjugating UBC6 and UBC7 and the Ub ligase HRD1 are the ER-associated Ub enzymes that mediate degradation of Sgk1; thus, Sgk1 has been identified as a cytosolic substrate for mammalian HRD1. Compartmentalization of Sgk1 controls the functional and spatial specificities of Sgk1-mediated signaling pathways, whereas rapid protein turnover provides a means to rapidly adjust Sgk1 abundance in response to different hormonal and external stimuli that increase Sgk1 gene transcription.

Animals↗
PubMed2006

Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways.

The yeast Doa10 ubiquitin (Ub) ligase resides in the endoplasmic reticulum (ER)/nuclear envelope (NE), where it functions in ER-associated degradation (ERAD). Doa10 substrates include non-ER proteins such as the transcription factor Mat alpha2. Here, we expand the range of Doa10 substrates to include a defective kinetochore component, a mutant NE membrane protein, and a substrate-regulated human ER enzyme. For all these substrates, Doa10 requires two Ub-conjugating enzymes, Ubc6 and Ubc7, as well as the Ubc7 cofactor Cue1. Based on a novel genomic screen of a comprehensive gene deletion library and other data, these four proteins appear to be the only nonessential and nonredundant factors generally required for Doa10-mediated ubiquitination. Notably, the Cdc48 ATPase facilitates degradation of membrane-embedded Doa10 substrates, but is not required for any tested soluble Doa10 substrates. This distinction is maintained even when comparing membrane and soluble proteins bearing the same degradation signal. Thus, while Doa10 ubiquitinates both membrane and soluble proteins, the mechanisms of subsequent proteasome targeting differ.

Cell Membrane↗
PubMed2006

Lingering mysteries of ubiquitin-chain assembly.

The small protein ubiquitin is often linked to substrates as a polymer. Such polymers vary in both linkage and length, which has important consequences for their function. Surprisingly, the mechanisms of ubiquitin-chain assembly are still not known. Deciphering them will shed light on why substrates differ in the extent and timing of polyubiquitin modification and how ancillary ubiquitination factors function.

Binding Sites↗
PubMed2006

The short-lived Matalpha2 transcriptional repressor is protected from degradation in vivo by interactions with its corepressors Tup1 and Ssn6.

The Matalpha2 (alpha2) protein is a transcriptional repressor necessary for the proper expression of cell type-specific genes in Saccharomyces cerevisiae. Like many transcription factors, alpha2 is rapidly degraded in vivo by the ubiquitin-proteasome pathway. At least two different ubiquitin-dependent pathways target alpha2 for destruction, one of which recognizes the well-characterized Deg1 degradation determinant near the N terminus of the protein. Here we report that the alpha2 corepressors Tup1 and Ssn6 modify the in vivo degradation rate of alpha2. Tup1 modulates the metabolic stability of alpha2 by directly binding to the Deg1-containing region of the protein. TUP1 overexpression specifically stabilizes Deg1-containing proteins but not other substrates of the same ubiquitination enzymes that recognize Deg1. Point mutations in both alpha2 and Tup1 that compromise the alpha2-Tup1 binding interaction disrupt the ability of Tup1 to stabilize Deg1 proteins. The physical association between Tup1 and alpha2 competes with the ubiquitination machinery for access to the Deg1 signal. Finally, we observe that overproduction of both Tup1 and Ssn6, but not either alone, strongly stabilizes the endogenous alpha2 protein. From these results, we propose that the fraction of alpha2 found in active regulatory complexes with Tup1 and Ssn6 is spared from rapid proteolytic destruction and is stabilized relative to the uncomplexed pool of the protein.

DNA-Binding Proteins↗
PubMed2006

Modification of proteins by ubiquitin and ubiquitin-like proteins.

Following the discovery of protein modification by the small, highly conserved ubiquitin polypeptide, a number of distinct ubiquitin-like proteins (Ubls) have been found to function as protein modifiers as well. These Ubls, which include SUMO, ISG15, Nedd8, and Atg8, function as critical regulators of many cellular processes, including transcription, DNA repair, signal transduction, autophagy, and cell-cycle control. A growing body of data also implicates the dysregulation of Ubl-substrate modification and mutations in the Ubl-conjugation machinery in the etiology and progression of a number of human diseases. The primary aim of this review is to summarize the latest developments in our understanding of the different Ubl-protein modification systems, including the shared and unique features of these related pathways.

Animals↗
PubMed2005

Membrane topology of the yeast endoplasmic reticulum-localized ubiquitin ligase Doa10 and comparison with its human ortholog TEB4 (MARCH-VI).

Quality control machinery in the endoplasmic reticulum (ER) helps ensure that only properly folded and assembled proteins accumulate in the ER or continue along the secretory pathway. Aberrant proteins are retrotranslocated to the cytosol and degraded by the proteasome, a process called ER-associated degradation. Doa10, a transmembrane protein of the ER/nuclear envelope, is one of the primary ubiquitin ligases (E3s) participating in ER-associated degradation in Saccharomyces cerevisiae. Here we report the membrane organization of the 1319-residue Doa10 polypeptide. The topology was determined by fusing a dual-topology reporter after 16 different Doa10 fragments. Our results indicate that Doa10 contains 14 transmembrane helices (TMs). Based on protease digestion of yeast microsomes, both the N-terminal RING-CH domain and the C terminus face the cytosol. Notably, the experimentally derived topology was not predicted correctly by any of the generally available TM prediction algorithms. Bioinformatic analysis and in silico mutagenesis guided the topological studies through problematic regions. The conserved TD domain in Doa10 includes three TMs. These TMs might function in cofactor binding or substrate recognition, or they might be part of a retrotranslocation channel. The Derlins were previously proposed to provide such channels, but we show that the two yeast Derlins are not required for degradation of Doa10 membrane substrates, as was found before for the Sec61 translocon. Finally, we provide evidence that the likely human Doa10 ortholog, TEB4 (MARCH-VI), adopts a topology similar to that of Doa10.

Algorithms↗
PubMed2005

Rtt106p is a histone chaperone involved in heterochromatin-mediated silencing.

Epigenetic inheritance of heterochromatin structure is an important cellular process whose mechanism remains elusive. In this article, we describe the identification of nine enhancers of the silencing defect of a Saccharomyces cerevisiae-PCNA mutant by screening a library of approximately 4,700 viable yeast deletion mutants. Of the nine mutants identified, six (hir1, hir3, sas2, sas4, sas5, and sir1) were previously known to reduce silencing synergistically with a mutation in Cac1p, the large subunit of chromatin assembly factor 1 (CAF-1). The predicted gene products that are affected in three other mutants (nam7, msh2, and rtt106) have not been implicated previously in silencing. Characterization of the rtt106Delta allele revealed that it synergistically reduced heterochromatin silencing when combined with a mutation in Cac1p but not with a mutation in Asf1p (a histone H3 and H4 chaperone). Moreover, Rtt106p interacted with histones H3 and H4 both in vitro and in vivo, and it displayed a nucleosome assembly activity in vitro. Furthermore, Rtt106p interacts with CAF-1 physically through Cac1p. These biochemical and genetic data indicate that Rtt106p is a previously uncharacterized histone chaperone connecting S phase to epigenetic inheritance.

Chromatin Assembly and Disassembly↗
PubMed2004

Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae.

SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo. Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes. However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways. Here we use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast. These three approaches yielded overlapping sets of proteins with the most extensive set by far being those identified by mass spectrometry. The two-hybrid data also yielded a potential SUMO-binding motif. Functional categories of SUMO-modified proteins include SUMO conjugation system enzymes, chromatin- and gene silencing-related factors, DNA repair and genome stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes. The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms.

Amino Acid Motifs↗
PubMed2004

Sem1, the yeast ortholog of a human BRCA2-binding protein, is a component of the proteasome regulatory particle that enhances proteasome stability.

Degradation of polyubiquitinated proteins by the proteasome often requires accessory factors; these include receptor proteins that bind both polyubiquitin chains and the regulatory particle of the proteasome. Overproduction of one such factor, Dsk2, is lethal in Saccharomyces cerevisiae and we show here that this lethality can be suppressed by mutations in SEM1, a gene previously recognized as an ortholog of the human gene encoding DSS1, which binds the BRCA2 DNA repair protein. Yeast sem1 mutants accumulate polyubiquitinated proteins, are defective for proteasome-mediated degradation and cannot grow under various stress conditions. Moreover, sem1 is synthetically lethal with mutations in proteasome subunits. We show that Sem1 is a component of the regulatory particle of the proteasome, specifically the lid subcomplex. Loss of Sem1 impairs the stability of the 26S proteasome and sem1Delta defects are greatly enhanced by simultaneous deletion of RPN10. The Rpn10 proteasome subunit appears to function with Sem1 in maintaining the association of the lid and base subcomplexes of the regulatory particle. Our data suggest a potential mechanism for this protein-protein stabilization and also suggest that an intact proteasomal regulatory particle is required for responses to DNA damage.

BRCA2 Protein↗
PubMed2004

Mechanism and function of deubiquitinating enzymes.

Attachment of ubiquitin to proteins is a crucial step in many cellular regulatory mechanisms and contributes to numerous biological processes, including embryonic development, the cell cycle, growth control, and prevention of neurodegeneration. In these diverse regulatory settings, the most widespread mechanism of ubiquitin action is probably in the context of protein degradation. Polyubiquitin attachment targets many intracellular proteins for degradation by the proteasome, and (mono)ubiquitination is often required for down-regulating plasma membrane proteins by targeting them to the vacuole (lysosome). Ubiquitin-protein conjugates are highly dynamic structures. While an array of enzymes directs the conjugation of ubiquitin to substrates, there are also dozens of deubiquitinating enzymes (DUBs) that can reverse the process. Several lines of evidence indicate that DUBs are important regulators of the ubiquitin system. These enzymes are responsible for processing inactive ubiquitin precursors, proofreading ubiquitin-protein conjugates, removing ubiquitin from cellular adducts, and keeping the 26S proteasome free of inhibitory ubiquitin chains. The present review focuses on recent discoveries that have led to a better understanding the mechanisms and physiological roles of this diverse and still poorly understood group of enzymes. We also discuss briefly some of the proteases that act on ubiquitin-like protein (UBL) conjugates and compare them to DUBs.

Amino Acid Sequence↗
PubMed2004

Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein.

The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.

Adenosine Triphosphatases↗
PubMed2004

Plasticity in eucaryotic 20S proteasome ring assembly revealed by a subunit deletion in yeast.

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/alpha3 subunit is deleted. Purified pre9Delta proteasomes show a two-fold enrichment for the Pre6/alpha4 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/alpha4 is expressed in yeast, it complements not only pre6Delta but also pre6Delta pre9Delta mutants; therefore, the plant alpha4 subunit also can occupy multiple positions in a functional yeast proteasome. Importantly, biogenesis of proteasomes is delayed at an early stage in pre9Delta cells, suggesting an advantage for Pre9 over Pre6 incorporation at the alpha3 position that facilitates correct assembly.

Arabidopsis↗