Intracellular protein degradation is required for maintaining the cellular proteome and regulating cellular processes. This pathway involves proximal ATP-dependent proteases that unfold and translocate proteins targeted for degradation into catalytic chambers. The large peptides produced are further cleaved by ATP independent endopeptidases, aminopeptidases and carboxypeptidases to release free amino acids. Lon and Clp are the key ATP-dependent proteases in prokaryotes and 26S proteasomes in eukayotes. In general, enzymes involved in the distal processing of peptides are ATP-independent, display greater redundancy and their orthologs are present in most organisms. The aim of the present study was to generate biochemical and functional insights on the ATP-independent enzyme, Peptidase N (PepN), which belongs to the M1 family. Previous studies in our laboratory identified Escherichia Coli PepN, to harbor both amino and endopeptidase activitities. In addition, it is responsible for the cleavage of majority of aminopeptidase substrates in E. Coli and is known to be involved in Sodium salicylate(NaSal)-induced stress. The present study consists of four parts. First, intracellular proteolysis plays an important role for virulence in pathogens. Therefore, it becomes important to study the biochemical properties and roles of enzymes involved in protein degradation. In this direction, a study was initiated to characterize the biochemical properties of Peptidase N from Salmonella enterica serovar Typhimurium(S. typhimurium). To study the contribution of PepN to the overall cystosolic protein degradation in S.typhimurium, a targeted deletion in pepN was generated. Cystosolic lysates of S. typhimurium wild type(WT) and ΔpepN strains were examined for their ability to cleave a panel of aminopeptidase and endopeptidase substrates. The ΔpepN strain displayed greatly reduced cleavage of nine out of a total of thirteen exopeptidase substrates, demonstrating a significant contribution of PepN to cytosolic aminopeptidase activity. S. typhimurium PepN also cleaved the endopeptidase substrate Suc-LLVY-AMC, similar to E. Coli PepN. To understand the physiological role of PepN, WT and ΔpepN were subjected to different stress conditions. During nutritional downshift in combination with high temperature stress, the growth of ΔpepN was significantly reduced compared to WT. Importantly, the PepN overexpressing strains grew better than WT, demonstrating an enhanced ability to overcome this stress combination. The above study clearly underscores the importance of PepN, to play distinct roles during stress. The significance of this study lies in understanding the biochemical and functional properties of a M1 family member from a pathogenic organism. Second, peptidases belonging to the M1 family are widely distributed with orthologs found across different kingdoms. The key amino acids in the catalytic domain are conserved in this family. However, amino acids present in the C-termini are variable and the three available crystal structures of M1 family members display distint differences in organization of this domain. To investigate the functional role of C-termini, progressive deletions were generated in PepN from E.Coli and Tricorn interacting factor F2 from Thermoplasma acidophilum(F2). Catalytic activity was partially reduced inPepN lacking four aa from C-terminus (PepNΔC4) whereas it is greatly reduced in F2 lacking ten amino acids from C-terminus(F2ΔC10) or eleven amino acids from PepN (PepNΔC11). To understand the mechanistic reasons involved, biochemical and biophysical studies were performed on purified WT and C-termini deleted proteins. Increased binding to 8-amino- 1- naphthalene sulphonic acid (ANS) was observed for all C-termini deleted proteins revealing greater numbers of surface exposed hydrophobic amino acids. Further, trypsin sensitivity studies demonstrated that mutant proteins were more sensitive compared to WT. Notably, expression of PepNΔC4, but not PepNΔC11, in E ColiΔpepN increased its ability to resist nutritional and high temperature stress, demonstrating a physiological role for the C-terminus. Together, these studies reveal involvement of distal amino acids in the C-termini of two distant M1 family members in repressing the exposure of apolar residues and enhancing enzyme function. Third, the crystal structure of E. coliPepN displayed the presence of Zn2+. To study the role of metal cofactor, apo-PepN was isolated by chelating the holoenzyme with 1,10-phenanthroline. Among different metals tested, only Zn2+ rescued the greatly reduced catalytic activity of the apo-PepN. Further confirmatory studies were performed using pepN mutants in the conserved GXMEN and HEXXH motifs. No major structural differences were observed in purified mutants(E264A, H297A, and E298A) using circular dichroism (CD) and intrinsic fluorescence studies; however, they lacked catalytic activity. These studies clearly demonstrate that Zn2+ was essential for catalysis but not for the overall structural integrity of PepN. Estimation of the Zn2+ content by atomic absorption spectrometry demonstrated that the WT contained one molecule of zinc per molecule of enzyme. Similar results were obtained in purified proteins of E264A and E298A. residues involved in catalysis. However the Zn2+ amount was greatly reduced in H297A, which is involved in Zn2+ binding. Further, the in vivo role of metal cofactor and catalyis were studied during two established stress conditions. Over expression of the mutants, unlike WT, was unable to rescue the growth of ΔpepN during nutritional down shift and high temperature stress. These results demonstrate that E264, H297 and E298 were required for PepN function during nutritional downshift and high temperature stress. However during NaSal-induced stress condition, overexpression of WT or mutants reduced growth of ΔpepN, demonstrating that PepN function was independent of catalytic activity or metal cofactor. Further studies identified the YL motif, which is conserved in all members of the M1 family, to play a role during NaSal-induced stress. Over expression of Y185F or L186Q did not modulate catalytic activity although growth reduction of ΔpepN in the presence of NaSal was compromised. To understand the mechanisms by which the YL motif plays a role during this condition, Y185F and L186Q mutant proteins were purified. In vitro, both mutant proteins were found to aggregate at a lower temperature and their catalytic activities were more sensitive to temperature, compared to WT. Steady state analysis of WT, Y185F and L186Q were performed to study the modulation of PepN amount during stress conditions. Steady state amounts of Y185F and L186Q mutant proteins were greatly decreased compared to WT, during NaSal-induced stress. Most likely, the lowered amounts of Y185F and L186Q mutant proteins contribute to growth advantage during NaSal-induced stress. Thus, the YL motif in E. Coli PepN reduces protein aggregation and enhances the structural integrity of PepN during selective stress conditions in vivo. In summary, this study clearly identifies metal cofactor and peptidase-dependent and –independent motifs to play distinct functional roles in PepN. Fourth, the crystal structures of known M1 family members have shown that the catalytic domain and mechanism of action are similar. To identify novel residues that may modulate the catalytic activity of PepN, multiple sequence alignment of important M1 family members were performed. The alignment identified a subset of M1 family members, including PepN, containing an aspargine residue which is present two amino acids before glycine in the GAMEN motif. A closer investigation of thecrystal structure of PepN revealed an interaction between N259(Catalytic domain) with Q821 (C-terminal domain). To understand the functional role of this interaction, site-specific mutants were generated: N259D, Q821E and a double mutant, N259D & Q821E. Spectroscopic studies did not reveal any significant differences with respect to global structure or protein stability between purified WT and mutant enzymes. Also, binding to substrates by mutant enzymes was not affected as judged by Km values. However, the Kcat of PepN containing N259D or Q821E was enhanced with respect to both aminopeptidase and endopeptidase substrates. On the other hand, there was significant decrease in the catalytic activity of the double mutant. Modeling studies demonstrate that the N259-Q821 interaction is located in the vicinity of residues important for catalysis in PepN and specific alterations in this interaction may affect the compactness of the catalytic domain. In summary, this study provides a functional role for the N259-Q821 interaction in modulating the catalytic activity of PepN. Mammalian orthologs of M1 family members play important roles in different physiological processes, e.g. angiogenesis, blood pressure, inflammation, MHC class I antigen presentation etc. PepN is a well characterized M1 family member of microbial origin. The present study on E. Coli PepN provides new knowledge on the roles of: a) distal C-terminal amino acids in repressing exposed hydrophobic amino acids; b) the conserved YL motif during NaSal-induced stress condition; c) the N259 and Q821 interaction in modulating enzymatic activity. The implications of these results on other members of the M1 family are discussed.

The aryl hydrocarbon receptor (Ahr) and the aryl hydrocarbon receptor nuclear translocator (Arnt) are well characterized bHLH-PAS transcription factors shown to regulate expression of xenobiotic metabolism genes. Extensive study has shown that upon treatment with certain aromatic hydrocarbons, mammalian cells rapidly activate the Ahr signaling pathway in order to stimulate gene expression and attempt to metabolize the xenobiotic compounds. It has been shown that after DNA-binding, the Ahr but not the Arnt protein, is quickly eliminated from the nuclear compartment thereby attenuating the dose of gene regulation administered by the Ahr*Arnt transcription factor complex. Previous studies have implicated involvement of the 26S proteasome complex in the degradation process, but the exact identity of the intermediary proteins and/or ligases remains to be defined. Identification and characterization of the protein(s) involved in degrading the receptor is essential for understanding the signaling pathway in its entirety including the mechanism for regulating the genetic response to Ahr ligands. The model organism, Saccharomyces cerevisiae, was used in order to characterize the Ahr signaling pathway and degradation mechanism in a more simplified cellular setting in which the major processes required for growth and development are conserved. First, the AHR and ARNT cDNAs were stably inserted into the yeast genome such that protein expression was inducible. A time course of induction demonstrated detectable levels of Ahr and Arnt proteins via western blotting while protein function was confirmed by detection of ligand-dependent reporter activity in an expressor strain carrying the pLXRE5-Z beta-galactosidase reporter plasmid. Additionally, a rapid reduction in protein levels was observed upon turning off the inducible GAL1 promoter located upstream of both AHR and ARNT cDNAs. Studies in mammalian cell culture have demonstrated that disrupting receptor chaperoning results in rapid Ahr protein turnover, as demonstrated by treatment with Hsp90 inhibitors. In order to determine if reduced Ahr protein expression in the yeast system was attributed to improper chaperoning of the exogenous protein; human heat shock proteins were constitutively expressed from yeast expression vectors in the Ahr and Arnt expressing strains, but did not confer any effect on Ahr stability when protein levels were evaluated by western blotting. Additionally, a strain of yeast was constructed such that the gene encoding the cell-wall protein, ERG6, was deleted from the yeast genome to allow for permeation of proteasome inhibitors. Treatment of this strain with proteasome inhibitors blocked the receptor degradation, therefore implicating the 26S proteasome in Ahr degradation when expressed exogenously in yeast.

Members of the forkhead class O (FOXO) transcription factors are tumor suppressors and key molecules that control aging and lifespan. The stability of mammalian FOXO proteins is controlled by proteasome-mediated degradation but general ubiquitin E3 ligases for FOXO factors remain to be defined. The current studies demonstrate that MDM2 bound to FOXO1 and FOXO3A and promoted their ubiquitination and subsequent degradation, a process apparently dependent on FOXO phopshorylation at PKB sites and on the E3 ligase activity of MDM2. The binding occurred between endogenous proteins and was involved the forkhead box of FOXO1 and the region of MDM2 that controls its cellular localization. MDM2 promoted the ubiquitination of FOXO1 in vitro in a cell free system. Knocking down MDM2 by siRNA caused the accumulation of endogenous FOXO3A protein, and enhanced the expression of FOXO target genes. In addition, MDM2 promoted the transcriptional activity of FOXO in a transient transfection system. In cells stably expressing a temperature sensitive mutant p53, activation of p53, by shifting to permissive temperatures led to MDM2 induction and the degradation of endogenous FOXO3A. These data suggested that MDM2 acts downstream of p53 as an E3 ubiquitin ligase to promote the degradation of mammalian FOXO factors.

Levels of macromolecules fluctuate both spatially and temporally in individual cells. Such heterogeneity could be exploited for bet hedging in uncertain environments, or be suppressed by negative feedback if perturbations are deleterious. For the master stress-response regulator in Escherichia coli, RpoS, both of these scenarios have been suggested. RpoS levels are also exceedingly low and controlled by the ClpXP protease, which reportedly displays extreme spatial heterogeneity. However, little is known quantitatively about RpoS dynamics. This is partly because no functional protein fusions exist, but also because the quantitative tools for studying fluctuations and localizations are limited, particularly ones that can be independently validated. Here I develop such methods and begin applying them to RpoS. Protein localization measurements increasingly rely on fluorescent protein fusions and are difficult to verify independently. I designed a non-intrusive method for validating localization patterns in live bacterial cells by exploiting post-division heterogeneity in downstream processes. Applying this assay to the ClpXP protease, widely reported to form biologically relevant foci, revealed in fact that the protease molecules are not specifically localized inside cells, as confirmed by four independent methods. I further evaluated 20+ commonly used fluorescent reporters and found that many cause severe mislocalization when fused to homo-oligomers, likely due to avidity effects. Further reinvestigating other foci-forming proteins strongly suggests that the previously reported foci were all caused by the fluorescent proteins used. For mRNAs – which are often present in low numbers per cell and major sources of non-genetic heterogeneity – existing single-cell assays have unknown accuracy: the experimental counting errors could completely over-shadow the natural variation. I therefore optimized and cross-evaluated two single-molecule mRNA detection methods. Several problems were identified and solutions discussed. I succeeded in building a functional RpoS protein fusion, and used bulk methods to show that the RpoS feedback loop is effectively not operating during exponential- phase growth. Mathematical analyses and initial experiments in a microfluidic device further suggest that the RpoS system has several unusual properties contributing towards extremely fast stress response. A stochastic analysis further suggests that the RpoS feedback loop cannot suppress spontaneous fluctuations, and preliminary experiments indicate that large deviations might indeed play important roles.

Deoxyribonucleic acid (DNA) carries all the genetic information of a cell. Ribonucleotide reductase (RNR) provides balanced pools of all four dNTPs, the building blocks of DNA. These building blocks are needed during DNA synthesis and repair. A failure in the control of the dNTP levels and/or their relative amounts leads to cell death or genetic abnormalities. Because of its central role in dNTP metabolism, RNR is highly regulated on multiple levels. The active RNR enzyme consists of two non-identical subunits called proteins R1 and R2. In mammalian cells, during an unperturbed cell cycle, the activity of RNR is highest during S and G2 phases. This is achieved by de novo synthesis of the limiting R2 protein at the onset of S phase, and by controlled degradation of the R2 protein during mitosis. This thesis deals with both the S phase-specific transcription of the mouse R2 gene, and the M phase-specific degradation of the mouse R2 protein. Sequence comparison of the mouse R2 promoter to human and guinea pig R2 promoters revealed some conserved elements. These putative regulatory elements were tested in both in vitro and in vivo transcription assays. We demonstrated that the previously identified, NF-Y binding CCAAT box is essential for high-level expression from the R2 promoter, but not for its S phase specificity. In addition, the conserved TATA box is dispensable both for basal and S phase-specific R2 transcription as long as the first 17 basepairs of the 5’ untranslated region are present. However, if this 5’ untranslated region is absent, the TATA box is needed for correct initiation of transcription. Focusing on the S phase specificity of the R2 gene expression, we demonstrated that the S phase-specific activity of the mouse R2 promoter is dependent on a protein-binding region located ~500 basepairs upstream of the transcription start site and an E2F binding site close to the transcription start site. Deletion of the upstream activating region results in an inactive promoter. In contrast, mutation of the E2F site leads to premature promoter activation in G1 and increased overall promoter activity. However, if the activating mutation of the E2F site is combined with mutation of the upstream activating region, the promoter becomes inactive. These results suggest that the E2F-dependent regulation is important but not sufficient for cell-cycle specific R2 transcription, and that the upstream activating region is crucial for the overall R2 promoter activity. In our studies of the M phase-specific R2 degradation, we found that it is dependent on a KEN sequence in the N-terminus of the R2 protein, recognized by the Cdh1-APC complex. Mutating the KEN box stabilizes the R2 protein during mitosis and G1 phase. In summary, these studies further extend our understanding of the regulation of the limiting R2 subunit of the enzyme ribonucleotide reductase. The S phase-specific transcription of the R2 gene and the M phase-specific degradation of the R2 protein may serve as important mechanisms to protect the cell against unscheduled DNA synthesis.

Seed formation consists of two major stages: embryo pattern formation and maturation. During seed maturation, the embryo accumulates storage material, acquires desiccation tolerance, and enters a stage of dormancy. Genetic analyses have identified several master regulators that orchestrate late embryogenesis, including the B3-domain transcription factor FUSCA3 (FUS3). In Arabidopsis, FUS3 has been shown to be a central regulator of hormonal pathways; it positively regulates late embryogenesis by increasing abscisic acid (ABA) level while repressing gibberellin (GA) synthesis. In turn, FUS3 protein level is positively and negatively regulated by ABA and GA, respectively. However, the mechanism of how this regulation occurs has not been well characterized. In this study, FUS3 has been shown to be an unstable protein rapidly degraded by the proteasome through a PEST instablility motif. To further characterize the mechanisms involved in FUS3 homeostasis, FUS3-interacting proteins were identified. The SnRK1 kinase AKIN10 was shown to interact with and phosphorylate FUS3 at its N-terminus. Furthermore, overexpression of AKIN10 delays FUS3 degradation, suggesting AKIN10 positively regulates FUS3 protein accumulation. Overexpression of AKIN10 delays developmental phase transitions, and causes defects in lateral organ development. These defects were partially rescued by the loss-of-function fus3-3 mutation, suggesting FUS3 and AKIN10 genetically interact to regulate these developmental processes. SnRK1/AMPK/Snf1 kinases are regulators of energetic stress responses. Overexpression studies suggest both FUS3 and AKIN10 positively regulate ABA signaling, but differ in sugar responses during germination; AKIN10 mediates glucose sensitivity, while FUS3 regulates osmotic stress responses. Overexpression of AKIN10 and FUS3 results in glucose and osmotic stress hypersensitivities, respectively, both of which are partially dependent on de novo ABA synthesis. Thus, FUS3 and AKIN10 act in overlapping pathways and combine different environmental signals to generate a common ABA-dependent response. In summary, novel mechanisms that regulate FUS3 homeostasis and function were identified. A model explaining the interaction between FUS3 and AKIN10 during embryonic and vegetative development, and the function of these two central developmental regulators in hormonal and stress signaling pathways is discussed.

Seed formation consists of two major stages: embryo pattern formation and maturation. During seed maturation, the embryo accumulates storage material, acquires desiccation tolerance, and enters a stage of dormancy. Genetic analyses have identified several master regulators that orchestrate late embryogenesis, including the B3-domain transcription factor FUSCA3 (FUS3). In Arabidopsis, FUS3 has been shown to be a central regulator of hormonal pathways; it positively regulates late embryogenesis by increasing abscisic acid (ABA) level while repressing gibberellin (GA) synthesis. In turn, FUS3 protein level is positively and negatively regulated by ABA and GA, respectively. However, the mechanism of how this regulation occurs has not been well characterized. In this study, FUS3 has been shown to be an unstable protein rapidly degraded by the proteasome through a PEST instablility motif. To further characterize the mechanisms involved in FUS3 homeostasis, FUS3-interacting proteins were identified. The SnRK1 kinase AKIN10 was shown to interact with and phosphorylate FUS3 at its N-terminus. Furthermore, overexpression of AKIN10 delays FUS3 degradation, suggesting AKIN10 positively regulates FUS3 protein accumulation. Overexpression of AKIN10 delays developmental phase transitions, and causes defects in lateral organ development. These defects were partially rescued by the loss-of-function fus3-3 mutation, suggesting FUS3 and AKIN10 genetically interact to regulate these developmental processes. SnRK1/AMPK/Snf1 kinases are regulators of energetic stress responses. Overexpression studies suggest both FUS3 and AKIN10 positively regulate ABA signaling, but differ in sugar responses during germination; AKIN10 mediates glucose sensitivity, while FUS3 regulates osmotic stress responses. Overexpression of AKIN10 and FUS3 results in glucose and osmotic stress hypersensitivities, respectively, both of which are partially dependent on de novo ABA synthesis. Thus, FUS3 and AKIN10 act in overlapping pathways and combine different environmental signals to generate a common ABA-dependent response. In summary, novel mechanisms that regulate FUS3 homeostasis and function were identified. A model explaining the interaction between FUS3 and AKIN10 during embryonic and vegetative development, and the function of these two central developmental regulators in hormonal and stress signaling pathways is discussed.

O típico sabor de chocolate é único, obtido somente de sementes fermentadas, secas e torradas de cacau, não podendo ser sintetizado artificialmente. O desenvolvimento desse sabor é influenciado pela constituição genética das sementes, processamento pós-colheita e manufatura. Proteínas dos cotilédones são potencialmente precursores do sabor e aroma de chocolate. O presente trabalho teve como objetivo analisar diferenças qualitativas e quantitativas nas proteínas de sementes de três genótipos de Theobroma cacao após a colheita e durante a fermentação, de forma a correlacionar estes resultados com diferenças na qualidade (sabor e aroma) obtidas por análise sensorial. Um dos desafios foi o isolamento de proteínas das sementes, evitando o alto teor de polifenóis e polissacarídeos que interferem na separação das proteínas e na análise do proteoma. A metodologia de extração composta por filtração em Miracloth, solubilização e precipitação em ácido ticloroacético (TCA) apresentou géis de maior resolução e repetibilidade, tendo sido escolhida como metodologia de extração protéica para estudo das alterações no proteoma das sementes de cacau durante a fermentação. Foi necessária também a utilização de kit comercial de purificação de proteínas e utilização de método de coloração com nitrato de prata para garantir géis com resolução dos spots e repetibilidade satisfatórias. Os spots foram isolados e após digestão tríptica, submetidos ao sequenciamento por cromatografia líquida associado ao espectrômetro de massas. Os espectros foram analisados pelo programa MASCOT MS/MS Ion Search, utilizando bancos de dados do NCBI. Análises dos mapas 2-D mostraram variação no número de spots entre as variedades. Ao final da fermentação, as proteínas ainda presentes nas sementes das variedades SIAL 70 e Catongo eram ácidas, e o processo de degradação foi caracterizado pelo desaparecimento de quase todas as proteínas neutras ou básicas e também de algumas proteínas ácidas; as proteínas com massa molar acima de 35 kDa também foram todas degradadas. Na variedade CCN 51, não ocorreu o mesmo perfil degradativo, havendo proteínas até pI 6,5 e massa molar acima de 100 kDa. Dos cem spots submetidos ao sequenciamento, 89 foram identificados. As proteínas 21kDa e vicilina foram as proteínas mais abundantes nos cotilédones. Correlacionando os resultados da análise sensorial e a proteômica concluiu-se que existe correlação tanto quantitativa como qualitativa das proteínas dos cotilédones de cacau e possivelmente com as proteínas precursoras de sabor de chocolate / Typical chocolate flavor is unique, only obtained from fermented, dried and roasted cocoa seeds, and can not be synthesized artificially. The flavor development is influenced by the genetic constitution, post-harvest processing and manufactures. Cotyledons proteins are believed to be the precursors of the chocolate flavor. The aim of the present work was to analyze qualitative and quantitative protein differences in seeds of three cocoa genotypes after harvesting and during the fermentation and to correlate these results with differences in quality (flavor and aroma) obtained by sensorial evaluation. One of the challenges was the isolation of proteins from cocoa seeds, avoiding the high content of polyphenols and polysaccharides which disturb protein separation and proteome analysis. The methodology of extraction by filtration in Miracloth, solubilization and precipitation in trichloroacetic acid showed the highest gel resolution and reprodutivity, and, thus, was chosen to be used in the analyses of the proteome of cocoa seeds during the fermentation. It was also necessary to use commercial kit for protein purification and a silver-based staining method with nitrate to guarantee gels with spots resolution and satisfactory reproducibility. Proteins were excised from de gels and after tryptic digestion, MS analysis was conducted by on line chromatografhy using a Cap-LC coupled to a mass spectrometer. The spectra were processed using MASCOT MS/MS Ion Search, and the sequences searched against NCBI databases. The 2-DE maps analysis of cocoa seeds showed significant variation of the spots number among the genotypes. At the end of fermentation, proteins still present in the Sial 70 and Catongo genotypes were acid and the degradation process was characterized by the disappearance of almost all the neutral or basic proteins and also some acid proteins. The genotype CCN 51 did not show the same degradation profile. Of the spots submitted to the mass spectrometer, 89 were identified. The 21kDa protein and vicilin were the most abundant proteins in the cocoa cotyledons. Correlating sensorial analysis and the proteomic results we could observe the existence of quantitative as qualitative correlation of proteins from cocoa cotyledons and possibly with the precursors proteins of chocolate flavor

Mitochondrial protein biogenesis depends on the import of nucleus-encoded precursors from the cytosol. Import is highly regulated and specific for different subcompartments, with intermembrane space (IMS) import driven by an oxidative mechanism on conserved cysteine residues. Oxidative folding in the IMS is facilitated by the mitochondria import and assembly (MIA) pathway. Proteins can only be imported into the IMS in Cys-reduced unfolded forms, as oxidation prevents translocation into the IMS. How the import-competent forms are maintained in the cytoplasm is lesser characterised compared to the MIA pathway. Two recent studies suggest that the cytosolic Thioredoxin (Trx) and Glutaredoxin (Grx) reductase systems play a role in facilitating IMS protein import, with previous evidence identifying a role for yeast Trxs in small Tim protein biogenesis. In this study, the redox properties of the yeast Trx and Grx systems were investigated, as well as whether the yeast Grx system play a role in the biogenesis of two typical types of IMS precursor proteins. First, in vitro studies were carried out to determine the standard redox potentials (E°’) of the Trx and Grx enzymes. This was a quantifiable parameter of reducing activity and the results were described in Chapter 3. This study determined the E°’Trx1 value, which was shown to be a more effective reductant compared to other orthologs. Experimental limitations prevented the Grx system E°’ values being determined. Next, whether the Grx plays a role in the biogenesis of the CX3C motif-containing small Tim proteins were investigated using yeast genetic in vivo and biochemical analysis methods. The results were described in Chapter 4. There, Grxs were observed to not affect cell growth, but in using overexpressed Tim9 as an import model, significant differences were observed for the Grx system as GRX deletion significantly decreased overexpressed Tim9 levels. Study on the isolated mitochondria and cytosol with overexpressed Tim9 was unclear however. This study also observed a genetic interaction between GRX andYME1 that recovered cell functioning under respiratory conditions. Finally, whether the Grx system plays a role in the biogenesis of CX9C motif-containing proteins (Mia40, Mia40C and Cox17) was studied in Chapter 5. Whilst Mia40C (C-domain of Mia40) and Cox17 are imported into mitochondria via the MIA pathway, the full-length Mia40 is a substrate of the presequence-targeted TIM23 pathway. The results indicated that import of the full-length Mia40 was unaffected by GRX deletion. However, studies of an overexpressed Mia40C as a substrate of the MIA pathway, showed strong mitochondrial protein level decreases caused by deletion of the Grx proteins. This decrease was also accompanied by an accumulation of unimported Mia40C in the cytosol. Cox17 as an alternative MIA pathway substrate also showed decreased mitochondrial levels in the GRX deletion mutants. These results altogether suggest that the cytosolic Grx system can function in the biogenesis of CX9C motif-containing IMS proteins imported through the MIA pathway, as well as the CX3C small Tim proteins. The topic of how IMS proteins are degraded in the cell was also raised by the study of Yme1.

A large fraction of the proteome displays cell cycle-dependent expression, which is important for cells to accurately grow and divide. Cyclical protein expression requires protein degradation via the ubiquitin proteasome system (UPS), and several ubiquitin ligases (E3) have established roles in this regulation. Less is understood about the roles of deubiquitinating enzymes (DUB), which antagonize E3 activity. A few DUBs have been shown to interact with and deubiquitinate cell cycle-regulatory E3s and their protein substrates, suggesting DUBs play key roles in cell cycle control. However, in vitro studies and characterization of individual DUB deletion strains in yeast suggest that these enzymes are highly redundant, making it difficult to identify their in vivo substrates and therefore fully understand their functions in the cell. To determine if DUBs play a role in the cell cycle, I performed a screen to identify specific DUB targets in vivo and then explored how these interactions contribute to cell cycle control. I conducted an in vivo overexpression screen to identify specific substrates of DUBs from a sample of UPS-regulated proteins and I determined that DUBs regulate different subsets of targets, confirming they display specificity in vivo. Five DUBs regulated the largest number of substrates, with Ubp10 stabilizing 40% of the proteins tested. Deletion of Ubp10 delayed the G1-S transition and reduced expression of Dbf4, a regulatory subunit of Cdc7 kinase, demonstrating Ubp10 is important for progression into S-phase. We hypothesized that compound deletion strains of these five DUBs would be deficient in key cellular processes because they regulated the largest number of cell cycle proteins from our screen. I performed genetic analysis to determine if redundancies exist between these DUBs. Our results indicate that most individual and combination deletion strains do not have impaired proliferation, with the exception of cells lacking UBP10. However, I observed negative interactions in some combinations when cells were challenged by different stressors. This implies the DUB network may activate redundant pathways only upon certain environmental conditions. While deletion of UBP10 impaired proliferation under standard growth conditions, I discovered that deletion of the proteasome-regulatory DUBs Ubp6 or Ubp14 rescues the cell cycle defect inubp10∆ cells. This suggests in the absence of Ubp10 substrates such as Dbf4 are rapidly degraded by the proteasome, but deletion of proteasome-associated DUBs restores cell cycle progression. Our work demonstrates that in unperturbed cells DUBs display specificity for their substrates in vivo and that a coordination of DUB activities promotes cell cycle progression.