In addition to apoptosis, metacapases function to regulate various other processes that promote and sustain life. For example, the Saccharomyces cerevisiae metacaspase Yca1 promotes cellular fitness by regulating insoluble protein levels. However, the mechanism(s) that regulate this proteostasis function for Yca1 have remained elusive. Here, using proteomics coupled to protein interaction studies, we describe a role for Yca1 in restraining deposition to the insoluble proteome and further identify a post-translational regulatory mechanism for modulating Yca1 function. Our initial analyses uncovered a role for Yca1 in aggregate assembly where Yca1, in coordination with the Cdc48 chaperone, regulates the composition of the insoluble proteome. Interestingly, loss of Yca1 was correlated with reduced sequestration of proteins related to ribosomal and translational processes in the insoluble protein fraction during heat stress. Subsequent proteomic analyses identified a regulatory mechanism for Yca1 mediated by the ubiquitin system, a feature that was instrumental for limiting insoluble protein content. Specifically, we noted K355 ubiquitination and S346 phosphorylation as key modifications that directed Yca1 function to maintain proteostasis. Loss of function mutations at these sites led to increased retention of insoluble protein and increased vacuolar structures. Surprisingly, loss of Yca1 also affected ubiquitin homeostasis in vivo as observed by reduced levels of low molecular weight free ubiquitin. Upon further analysis, we observed that the ubiquitin precursor protein Rsp31 was cleaved by Yca1 suggesting a possible role for Yca1 in de novo ubiquitin synthesis. Together, these analyses suggest that post-translational modifications of Yca1 are critical regulatory features for this protease, and that this enzyme regulates cell proteostasis in combination with other chaperone and protein degradation machinery.

Ubiquitination is a post-translational modification that can take several forms. Some proteins are modified with a single ubiquitin molecule, while others are modified with polyubiquitin chains. Each type of ubiquitination is thought to have distinct biological functions. The best-characterized types of ubiquitin modification are K48-linked polyubiquitination, which serves as a signal for proteasomal degradation and K63-linked polyubiquitination, which has non-proteolytic functions such in DNA repair, signaling, and endocytosis. HECT ubiquitin ligases (HECT E3s) form a class of E3s, defined by a C terminal catalytic domain. Several lines of evidence suggested that the HECT E3s assemble a polyubiquitin chain in a sequential manner with one molecule of ubiquitin at a time being conjugated to the distal ubiquitin of the chain. In the process of chain elongation, not all HECT E3s target a common internal lysine of ubiquitin, leading to diversification of chain type specificity in HECT E3s. For example, yeast Rsp5 forms K63 chains, while human E6AP forms K48 chains. Two important mechanistic questions were addressed in my work: 1) what are the determinants of chain type specificity of HECT E3s, and 2) what allows the distal ubiquitin of a chain to be continuously oriented near the active site of the HECT domain in the course of a sequential polyubiquitination reaction? I have determined that the chain type specificity of Rsp5 is a function solely of the HECT domain. Further, through the generation of chimeric HECT E3s, I demonstrated that chain type specificity determinants are located within the last 60 amino acids of the C lobe of the HECT domain. To address the second question, we solved the structure of Rsp5 HECT domain in complex with non-covalently bound ubiquitin in collaboration with Jue Chen’s laboratory (Purdue University). From the structure, we found that the N lobe of the HECT domain binds ubiquitin in a manner distinct from other known ubiquitin binding domains, and I have shown that Rsp5 proteins defective for ubiquitin binding are defective for chain elongation. We hypothesize that the ubiquitin binding site functions in the recruitment of the distal ubiquitin of polyubiquitin chain for efficient polyubiquitination. / text

Ubiquitylation is an indispensable post-translational modification system in eukaryotic cells that leads to the covalent attachment of a small ubiquitin (Ub) protein onto a target. The traditional and best-characterized role for ubiquitylation is a fundamental regulatory mechanism whereby target proteins are tagged with a characteristic Lys48-linked Ub chain that signals for their elimination through proteasomal degradation. Challenging this conventional wisdom is the finding that some ubiquitylated proteins are modified by Ub chains linked through Lys63, providing a molecular signal that is thought to be structurally and functionally distinct from Lys48-linked Ub chains. Of further interest and significance is that the Lys63-linked Ub chains are apparently synthesized through a novel biochemical mechanism employing a unique complex formed between a true Ub conjugating enzyme (E2), Ubc13, and an E2-variant (Uev), Mms2 (or Uev1A). The goal of this thesis was to employ structural and functional approaches in order to better characterize the Ubc13-Mms2 Lys63-linked Ub conjugation complex. <p>Error-free DNA damage tolerance (DDT) in the budding yeast is dependent on Lys63-linked Ub chains synthesized by Ubc13-Mms2 and thus provided the opportunity to experimentally test the function of the human UBC13 and MMS2 genes in a simple model organism. Human UBC13 and MMS2 were each shown to function in place of their yeast counterparts and in accordance, human Ubc13 was shown to physically interact with yeast Mms2, and vice versa. Two human MMS2 homologs were also tested and it was determined that UEV1A but not UEV1B can function in place of mms2 in yeast DDT. Physical interactions were observed between Ubc13 and Uev1A, but not between Ubc13 and Uev1B, suggesting that Ubc13-Uev complex formation is required for function. <p>In collaboration with a research group at the University of Alberta, crystal structure and NMR data were used to develop a mechanistic model for the conjugation of Lys63-linked Ub chains by the Ubc13-Mms2 heterodimer, whereby the special orientation of two Ub molecules facilitates a specific Ub-Ub linkage via Lys63. In order to help support the in vitro model and to determine how the Ubc13-Mms2 structure relates to biological function, I used a structure-based approach to direct the creation of point mutations within four key regions of the Ubc13-Mms2 heterodimer; the Ubc13 active-site, the Ubc13-E3 (Ub ligating enzyme) interface, the Mms2-Ub interface, and the Ubc13-Mms2 interface. <p>Underscoring the importance of the Ub conjugation by Ubc13-Mms2, a Ubc13-C87S active-site mutation was created that could bind to Mms2 but was unable to function in DDT. Regarding the Ubc13-E3 interface, a single Ubc13-M64A point mutation had a potent effect on disrupting Ubc13 function in DDT, as well as its physical interaction with Rad5, TRAF6, and CHFR. The results suggest that different RING finger E3s use the same Ubc13 surface to sequester the Ub conjugation activity of Ubc13-Mms2. Two human Mms2 mutations at Ser32 and Ile62, which are contained within the Mms2-Ub interface, were found to reduce the ability of Mms2 to bind Ub. When the corresponding yeast mutations are combined, a synergistic loss in DDT function is observed. The relative orientation of Ser32 and Ile62 suggests that the Mms2 and Tsg101 Uev families use different Uev surfaces to physically interact with Ub. A 200 ìM dissociation constant for the wild-type Mms2-Ub interaction was also determined. The systematic mutagenesis and testing of 14 Ubc13-Mms2 interface residues led to mutants with partial or complete disruption of binding and function. Using this data, a model involving the insertion of a specific Mms2-Phe residue into a unique Ubc13 hydrophobic pocket was created to explain the specificity of Mms2 for Ubc13, and not other E2s. In addition, the dissociation constant for the wild-type Ubc13-Mms2 heterodimer was determined to be approximately 50 nM. <p>The structural and functional studies strongly support the notion that Ubc13-Mms2 complex has the unique ability to conjugate Lys63-linked Ub chains. However, several reported instances of Lys63-linked Ub chains in vivo have not yet been attributed to Ubc13 or Mms2. To address the disparity I was able to demonstrate and map a physical interaction between Mms2 and Rsp5, an E3 implicated in Lys63-linked Ub conjugation. Surprisingly, it was found that MMS2 is not responsible for the RSP5-dependent Lys63-linked Ub conjugation of a plasma membrane protein. A possible explanation for the apparent paradox is presented.

Ubiquitylation is an indispensable post-translational modification system in eukaryotic cells that leads to the covalent attachment of a small ubiquitin (Ub) protein onto a target. The traditional and best-characterized role for ubiquitylation is a fundamental regulatory mechanism whereby target proteins are tagged with a characteristic Lys48-linked Ub chain that signals for their elimination through proteasomal degradation. Challenging this conventional wisdom is the finding that some ubiquitylated proteins are modified by Ub chains linked through Lys63, providing a molecular signal that is thought to be structurally and functionally distinct from Lys48-linked Ub chains. Of further interest and significance is that the Lys63-linked Ub chains are apparently synthesized through a novel biochemical mechanism employing a unique complex formed between a true Ub conjugating enzyme (E2), Ubc13, and an E2-variant (Uev), Mms2 (or Uev1A). The goal of this thesis was to employ structural and functional approaches in order to better characterize the Ubc13-Mms2 Lys63-linked Ub conjugation complex. <p>Error-free DNA damage tolerance (DDT) in the budding yeast is dependent on Lys63-linked Ub chains synthesized by Ubc13-Mms2 and thus provided the opportunity to experimentally test the function of the human UBC13 and MMS2 genes in a simple model organism. Human UBC13 and MMS2 were each shown to function in place of their yeast counterparts and in accordance, human Ubc13 was shown to physically interact with yeast Mms2, and vice versa. Two human MMS2 homologs were also tested and it was determined that UEV1A but not UEV1B can function in place of mms2 in yeast DDT. Physical interactions were observed between Ubc13 and Uev1A, but not between Ubc13 and Uev1B, suggesting that Ubc13-Uev complex formation is required for function. <p>In collaboration with a research group at the University of Alberta, crystal structure and NMR data were used to develop a mechanistic model for the conjugation of Lys63-linked Ub chains by the Ubc13-Mms2 heterodimer, whereby the special orientation of two Ub molecules facilitates a specific Ub-Ub linkage via Lys63. In order to help support the in vitro model and to determine how the Ubc13-Mms2 structure relates to biological function, I used a structure-based approach to direct the creation of point mutations within four key regions of the Ubc13-Mms2 heterodimer; the Ubc13 active-site, the Ubc13-E3 (Ub ligating enzyme) interface, the Mms2-Ub interface, and the Ubc13-Mms2 interface. <p>Underscoring the importance of the Ub conjugation by Ubc13-Mms2, a Ubc13-C87S active-site mutation was created that could bind to Mms2 but was unable to function in DDT. Regarding the Ubc13-E3 interface, a single Ubc13-M64A point mutation had a potent effect on disrupting Ubc13 function in DDT, as well as its physical interaction with Rad5, TRAF6, and CHFR. The results suggest that different RING finger E3s use the same Ubc13 surface to sequester the Ub conjugation activity of Ubc13-Mms2. Two human Mms2 mutations at Ser32 and Ile62, which are contained within the Mms2-Ub interface, were found to reduce the ability of Mms2 to bind Ub. When the corresponding yeast mutations are combined, a synergistic loss in DDT function is observed. The relative orientation of Ser32 and Ile62 suggests that the Mms2 and Tsg101 Uev families use different Uev surfaces to physically interact with Ub. A 200 ìM dissociation constant for the wild-type Mms2-Ub interaction was also determined. The systematic mutagenesis and testing of 14 Ubc13-Mms2 interface residues led to mutants with partial or complete disruption of binding and function. Using this data, a model involving the insertion of a specific Mms2-Phe residue into a unique Ubc13 hydrophobic pocket was created to explain the specificity of Mms2 for Ubc13, and not other E2s. In addition, the dissociation constant for the wild-type Ubc13-Mms2 heterodimer was determined to be approximately 50 nM. <p>The structural and functional studies strongly support the notion that Ubc13-Mms2 complex has the unique ability to conjugate Lys63-linked Ub chains. However, several reported instances of Lys63-linked Ub chains in vivo have not yet been attributed to Ubc13 or Mms2. To address the disparity I was able to demonstrate and map a physical interaction between Mms2 and Rsp5, an E3 implicated in Lys63-linked Ub conjugation. Surprisingly, it was found that MMS2 is not responsible for the RSP5-dependent Lys63-linked Ub conjugation of a plasma membrane protein. A possible explanation for the apparent paradox is presented.