The Ulp1 SUMO isopeptidase: distinct domains required for viability, nuclear envelope localization, and substrate specificity

Protein modification by the ubiquitin-like SUMO protein contributes to many cellular regulatory mechanisms. In Saccharomyces cerevisiae, both sumoylating and desumoylating activities are essential for viability. Of its two known desumoylating enzymes, Ubl-specific protease (Ulp)1 and Ulp2/Smt4, Ulp1 is specifically required for cell cycle progression. A ∼200-residue segment, the Ulp domain (UD), is conserved among Ulps and includes a core cysteine protease domain that is even more widespread. Here we demonstrate that the Ulp1 UD by itself can support wild-type growth rates and in vitro can cleave SUMO from substrates. However, in cells expressing only the UD of Ulp1, many SUMO conjugates accumulate to high levels, indicating that the nonessential Ulp1 NH2-terminal domain is important for activity against a substantial fraction of sumoylated targets. The NH2-terminal domain also includes sequences necessary and sufficient to concentrate Ulp1 at nuclear envelope sites. Remarkably, NH2-terminally deleted Ulp1 variants are able, unlike full-length Ulp1, to suppress defects of cells lacking the divergent Ulp2 isopeptidase. Thus, the NH2-terminal regulatory domain of Ulp1 restricts Ulp1 activity toward certain sumoylated proteins while enabling the cleavage of others. These data define key functional elements of Ulp1 and strongly suggest that subcellular localization is a physiologically significant constraint on SUMO isopeptidase specificity

Characterization and analysis of genes encoding Escherichia coli acetyl-CoA carboxylase

Acetyl-CoA carboxylase catalyzes the first committed step of fatty acid biosynthesis, the synthesis of malonyl-CoA. In Escherichia coli the enzyme is a complex of four subunits that catalyzes two distinct half-reactions.I have cloned and determined the nucleotide sequences of the genes encoding biotin carboxylase, BCCP (map at min 72), and carboxyltransferase subunits (The $\alpha$ subunit map at min 4.3 and the $\beta$ subunit map at min 50). I have also defined the sequence requirement for biotination of the BCCP subunit by protein fusion analyses. Peptide mapping of the purified carboxyltransferase indicates that this enzyme component is a complex of two nonidentical subunits. In addition, I have identified putative functional domains within the deduced amino acid sequences. The identified domains include sequences that involves in ATP, bicarbonate binding (biotin carboxylase), in biotination (BCCP), and in CoA binding (carboxyltransferase).To investigate the regulation of acetyl-CoA carboxylase genes, I monitored the transcription levels of these genes under a variety of growth conditions. A direct correlation was found between the levels of acc genes transcripts and the rate of cellular growth.U of I OnlyETDs are only available to UIUC Users without author permissio

The Saccharomyces cerevisiae ubiquitin-proteasome system

Our studies of the yeast ubiquitin-proteasome pathway have uncovered a number of general principles that govern substrate selectivity and proteolysis in this complex system. Much of the work has focused on the destruction of a yeast transcription factor, MAT alpha 2. The alpha 2 protein is polyubiquitinated and rapidly degraded in alpha-haploid cells. One pathway of proteolytic targeting, which depends on two distinct endoplasmic reticulum-localized ubiquitin-conjugating enzymes, recognizes the hydrophobic face of an amphipathic helix in alpha 2. Interestingly, degradation of alpha 2 is blocked in a/alpha-diploid cells by heterodimer formation between the alpha 2 and a1 homeodomain proteins. The data suggest that degradation signals may overlap protein-protein interaction surfaces, allowing a straightforward steric mechanism for regulated degradation. Analysis of alpha 2 degradation led to the identification of both 20S and 26S proteasome subunits, and several key features of proteasome assembly and active-site formation were subsequently uncovered. Finally, it has become clear that protein (poly) ubiquitination is highly dynamic in vivo, and our studies of yeast de-ubiquitinating enzymes illustrate how such enzymes can facilitate the proteolysis of diverse substrates