Proteomic analysis of the Cdc48/Ubx network identifies a role for Ubx2 in the regulation of lipid biosynthesis

Cdc48/p97 is an essential, highly abundant hexameric member of the AAA (ATPase associated with various cellular activities) family. It has been linked to a variety of processes throughout the cell but it is best known for its role in the ubiquitin proteasome pathway. In this system it is believed that Cdc48 behaves as a segregase, transducing the chemical energy of ATP hydrolysis into mechanical force to separate ubiquitin-conjugated proteins from their tightly-bound partners.

Current models posit that Cdc48 is linked to its substrates through a variety of adaptor proteins, including a family of seven proteins (13 in humans) that contain a Cdc48-binding UBX domain. As such, due to the complexity of the network of adaptor proteins for which it serves as the hub, Cdc48/p97 has the potential to exert a profound influence on the ubiquitin proteasome pathway. However, the number of known substrates of Cdc48/p97 remains relatively small, and smaller still is the number of substrates that have been linked to a specific UBX domain protein. As such, the goal of this dissertation research has been to discover new substrates and better understand the functions of the Cdc48 network. With this objective in mind, we established a proteomic screen to assemble a catalog of candidate substrate/targets of the Ubx adaptor system.

Here we describe the implementation and optimization of a cutting-edge quantitative mass spectrometry method to measure relative changes in the Saccharomyces cerevisiae proteome. Utilizing this technology, and in order to better understand the breadth of function of Cdc48 and its adaptors, we then performed a global screen to identify accumulating ubiquitin conjugates in cdc48-3 and ubxΔ mutants. In this screen different ubx mutants exhibited reproducible patterns of conjugate accumulation that differed greatly from each other, pointing to various unexpected functional specializations of the individual Ubx proteins.

As validation of our mass spectrometry findings, we then examined in detail the endoplasmic-reticulum bound transcription factor Spt23, which we identified as a putative Ubx2 substrate. In these studies ubx2Δ cells were deficient in processing of Spt23 to its active p90 form, and in localizing p90 to the nucleus. Additionally, consistent with reduced processing of Spt23, ubx2Δ cells demonstrated a defect in expression of their target gene OLE1, a fatty acid desaturase. Overall, this work demonstrates the power of proteomics as a tool to identify new targets of various pathways and reveals Ubx2 as a key regulator lipid membrane biosynthesis.

Negative regulation of transcription factors by Srb10 cyclin-dependent kinase

The ubiquitin-dependent proteolytic pathway plays an important role in a broad array of cellular processes, inducting cell cycle control and transcription. Biochemical analysis of the ubiquitination of Sic1, the B-type cyclin-dependent kinase (CDK) inhibitor in budding yeast helped to define a ubiquitin ligase complex named SCF^cdc4 (for Skp1, Cdc53/cullin, F-box protein). We found that besides Sic1, the CDK inhibitor Far1 and the replication initiation protein Cdc6 are also substrates of SCF^cdc4 in vitro. A common feature in the ubiquitination of the cell cycle SCF^cdc4 substrates is that they must be phosphorylated by the major cell cycle CDK, Cdc28. Gcn4, a transcription activator involved in the general control of amino acid biosynthesis, is rapidly degraded in an SCF^cdc4-dependent manner in vivo. We have focused on this substrate to investigate the generality of the SCF^cdc4 pathway. Through biochemical fractionations, we found that the Srb10 CDK phosphorylates Gcn4 and thereby marks it for recognition by SCF^cdc4 ubiquitin ligase. Srb10 is a physiological regulator of Gcn4 stability because both phosphorylation and turnover of Gcn4 are diminished in srb10 mutants. Furthermore, we found that at least two different CDKs, Pho85 and Srb10, conspire to promote the rapid degradation of Gcn4 in vivo. The multistress response transcriptional regulator Msn2 is also a substrate for Srb10 and is hyperphosphorylated in an Srb10-dependent manner upon heat stress-induced translocation into the nucleus. Whereas Msn2 is cytoplasmic in resting wild type cells, its nuclear exclusion is partially compromised in srb10 mutant cells. Srb10 has been shown to repress a subset of genes in vivo, and has been proposed to inhibit transcription via phosphorylation of the C-terminal domain of RNA polymerase II. Our results suggest a general theme that Srb10 represses the transcription of specific genes by directly antagonizing the transcriptional activators.

I. Studies on alkaline phosphatase activity in developing sea urchin embryos. II. Studies on the activation of protein biosynthesis in sea urchin eggs at fertilization

I. Alkaline phosphatase activity in the developing sea urchin Lytechinus pictus has been investigated with respect to intensity at various stages, ionic requirements and intracellular localization. The activity per embryo remains the same in the unfertilized egg, fertilized egg and cleavage stages. At a time just prior to gastrulation (about 10 hours after fertilization) the activity per embryo begins to rise and increases after 300 times over the activity in the cleavage stages during the next 60 hours.

The optimum ionic strength for enzymatic activity shows a wide peak at 0.6 to 1.0. Calcium and magnesium show an additional optimum at a concentration in the range of 0.02 to 0.07 molar. EDTA at concentrations of 0.0001 molar and higher shows a definite inhibition of activity.

The intracellular localization of alkaline phosphatase in homogenates of 72-hour embryos has been studied employing the differential centrifugation method. The major portion of the total activity in these homogenates was found in mitochondrial and microsomal fractions with less than 5% in the nuclear fraction and less than 2% in the final supernatant. The activity could be released from all fractions by treatment with sodium deoxycholate.

II. The activation of protein biosynthesis at fertilization in eggs of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus has been studied in both intact eggs and cell-free homogenates. It is shown that homogenates from both unfertilized and fertilized eggs are dependent on potassium and magnesium ions for optimum amino acid incorporation activity and in the case of the latter the concentration range is quite narrow. Though the optimum magnesium concentrations appear to differ slightly in homogenates of unfertilized and fertilized eggs, in no case was it observed that unfertilized egg homogenates were stimulated to incorporate at a level comparable to that of the fertilized eggs.

An activation of amino acid incorporation into protein has also been shown to occur in parthenogenetically activated non-nucleate sea urchin egg fragments or homogenates thereof. This activation resembles that in the fertilized whole egg or fragment both in amount and pattern of activation. Furthermore, it is shown that polyribosomes form in these non-nucleate fragments upon artificial activation. These findings are discussed along with possible mechanisms for activation of the system at fertilization.