Cloning and characterization of Pros45, the Drosophila SUG1 proteasome subunit homolog

The proteasome plays essential roles in a variety of cellular processes, including degradation of the bulk of cellular proteins, degradation of short-lived proteins such as cell cycle regulators, generation of antigenic peptides, and mediating programmed cell death. One of the best characterized subunits of the 26S proteasome is encoded by the yeast gene SUG1. We report here the cloning and characterization of the Drosophila homolog of this gene, Pros45. At the protein level, Pros45 is highly conserved with respect to its homologs in a variety of taxa: it shows 74% identity to yeast Sug1; 86% to mouse m56/mSug1/FZA-B; 87% to human Trip1; and 97% to moth 18-56. Using a genomic clone as a probe for in situ hyridization to polytene chromesomes, we demonstrated that Pros45 maps to 19F, near the base of the X chromosome. Use of a pros45 cDNA clone as a probe revealed a second site of hybridization at 99CD. Pros45 mRNA is found in the unfertilized egg and in all cells of the early embryo. By the end of embryogenesis, Pros45 is expressed predominantly in the central nervous system. Targeted expression of Pros45 in a variety of different cells using the Gal4 UAS P-element system failed to generate an overt phenotype. This study provides the foundation for further examination of the role of the 26S proteasome in homeostasis and development in Drosophila. Received: 23 October 1997 / Accepted: 6 March 1998     

Cloning and characterization of Pros45, the Drosophila SUG1 proteasome subunit homolog

The proteasome plays essential roles in a variety of cellular processes, including degradation of the bulk of cellular proteins, degradation of short-lived proteins such as cell cycle regulators, generation of antigenic peptides, and mediating programmed cell death. One of the best characterized subunits of the 26S proteasome is encoded by the yeast gene SUG1. We report here the cloning and characterization of the Drosophila homolog of this gene, Pros45. At the protein level, Pros45 is highly conserved with respect to its homologs in a variety of taxa: it shows 74% identity to yeast Sug1; 86% to mouse m56/mSug1/FZA-B; 87% to human Trip1; and 97% to moth 18-56. Using a genomic clone as a probe for in situ hyridization to polytene chromesomes, we demonstrated that Pros45 maps to 19F, near the base of the X chromosome. Use of a pros45 cDNA clone as a probe revealed a second site of hybridization at 99CD. Pros45 mRNA is found in the unfertilized egg and in all cells of the early embryo. By the end of embryogenesis, Pros45 is expressed predominantly in the central nervous system. Targeted expression of Pros45 in a variety of different cells using the Gal4 UAS P-element system failed to generate an overt phenotype. This study provides the foundation for further examination of the role of the 26S proteasome in homeostasis and development in Drosophila.     

Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast.

The 26 S proteasome of eukaryotes is responsible for the degradation of proteins targeted for proteolysis by the ubiquitin system. Yeast has been an important model organism for understanding eukaryotic proteasome structure and function. Toward a quantitative characterization of the proteasome, we have determined the localization, cellular levels, and stoichiometry of proteasome subunits. The subcellular localization of two ATPase components of the regulatory complex of the proteasome, Sug2/Rpt4 and Sug1/Rpt6, and a subunit of the 20 S proteasome, Pre1, were determined by immunofluorescence. In contrast to findings in multicellular organisms, these proteins are localized almost exclusively to the nucleus throughout the cell cycle. We have also determined the cellular abundance and stoichiometry of these proteasome subunits. Sug1/Rpt6, Sug2/Rpt4, and Pre1 are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, corresponding to a concentration of 13-26 microM in the nucleus. Also, in contrast to mammalian cells, we find no evidence of a p27-containing "modulator" of the proteasome in yeast. This information will be useful in comparing and contrasting the yeast and mammalian proteasomes and should contribute to a mechanistic understanding of how this complex functions.     

MYO18B interacts with the proteasomal subunit Sug1 and is degraded by the ubiquitin-proteasome pathway

MYO18B is a class XVIIIB unconventional myosin encoded by a candidate tumor suppressor gene. To gain insights into the cellular function of this protein, we searched for MYO18B-interacting proteins by a yeast two-hybrid screen. Sug1, a 19S regulator subunit of the 26S proteasome, was identified as a binding partner of the C-terminal tail region of MYO18B. The association of MYO18B with Sug1 was further confirmed by GST pull-down, co-immunoprecipitation, and immunocytochemistry. Furthermore, proteasome dysfunction by a proteasome inhibitor or siRNA-mediated knock-down of Sug1 caused the up-regulation of MYO18B protein and MYO18B was polyubiquitinated in vivo. Collectively, these results suggested that MYO18B is a substrate for proteasomal degradation.     

Linkage mapping and fluorescence in situ hybridization of TCTE1 on human chromosome 6p: Analysis of dinucleotide polymorphisms on native gels

Highly informative dinucleotide repeat polymorphisms were identified at the T-complex-associated-testes-expressed-1 (TCTE1) locus on human chromosome 6p. Electrophoresis of single-stranded DNA on native gels facilitated the analysis of the dinucleotide polymorphisms. Linkage mapping positions this marker midway between the centromere and HLA with recombination fractions as follows: D6Z1-0.21-TCTE1-0.24-HLA. Two-color fluorescence in situ hybridization places TCTE1 proximal to CRIL171 (D6S19). Together, linkage and in situ hybridization indicate that the order of the loci is D6Z1-D6S4-D6S90-TCTE1-D6S19-D6S29-HLA-telomere. A sequence tagged site (STS) was established, and three yeast artificial chromosome (YAC) clones were identified for the TCTE1 locus.     

Isolation,characterization, and chromosomal mapping of a novel cDNA clone encoding human selenium binding protein

We have isolated the full-length human 56 kDa selenium binding protein (hSP56) cDNA clone, which is the human homolog of mouse 56 kDa selenium binding protein. The cDNA is 1,668 bp long and has an open reading frame encoding 472 amino acids. The calculated molecular weight is 52.25 kDa and the estimated isoelectric point is 6.13. Using Northern blot hybridization, we found that this 56 kDa selenium binding protein is expressed in mouse heart with an intermediate level between those found in liver/lung/kidney and intestine. We have also successfully expressed hSP56 in Escherichia coli using the expression vector-pAED4. The hSP56 gene is located at human chromosome 1q21–22. J. Cell. Biochem. 64:217–224. © 1997 Wiley-Liss, Inc.     

Co‐ and post‐translational modifications of the 26S proteasome in yeast

The yeast (Saccharomyces cerevisiae) 26S proteasome consists of the 19S regulatory particle (19S RP) and 20S proteasome subunits. We detected comprehensively co‐ and post‐translational modifications of these subunits using proteomic techniques. First, using MS/MS, we investigated the N‐terminal modifications of three 19S RP subunits, Rpt1, Rpn13, and Rpn15, which had been unclear, and found that the N‐terminus of Rpt1 is not modified, whereas that of Rpn13 and Rpn15 is acetylated. Second, we identified a total of 33 Ser/Thr phosphorylation sites in 15 subunits of the proteasome. The data obtained by us and other groups reveal that the 26S proteasome contains at least 88 phospho‐amino acids including 63 pSer, 23 pThr, and 2 pTyr residues. Dephosphorylation treatment of the 19S RP with λ phosphatase resulted in a 30% decrease in ATPase activity, demonstrating that phosphorylation is involved in the regulation of ATPase activity in the proteasome. Third, we tried to detect glycosylated subunits of the 26S proteasome. However, we identified neither N‐ and O‐linked oligosaccharides nor O‐linked β‐N‐acetylglucosamine in the 19S RP and 20S proteasome subunits. To date, a total of 110 co‐ and post‐translational modifications, including N^α‐acetylation, N^α‐myristoylation, and phosphorylation, in the yeast 26S proteasome have been identified.     

Characterization of the Mouse Src Homology 3 Domain GeneSh3d2con Chr 7 Demonstrates Coexpression with Huntingtin in the Brain and Identifies the Processed PseudogeneSh3d2c-ps1on Chr 2

Formation of intracellular protein complexes is often mediated by Src homology 3 domain-containing proteins interacting with proline-rich target sequences on other proteins. TheSh3d2cgene or its rat/human orthologs have been implicated in synaptic vesicle recycling due to interaction with dynamin I and synaptojanin in nerve terminals. In a yeast two-hybrid system, association with a huntingtin fragment containing an elongated stretch of polyglutamines was observed recently. By genetic mapping and fluorescencein situhybridization we demonstrate the localization ofSh3d2con mouse chromosome 7. A processed pseudogene ofSh3d2c, Sh3d2c-ps1,was identified and mapped to mouse chromosome 2. Using RNAin situhybridization, we show thatSh3d2cis transcribed in various regions of the brain. The striatum, hippocampus, cortex, basal hypothalamus, brain stem, and cerebellum are the most prominent sites of expression. Because huntingtin andSh3d2care coexpressed in most regions of the brain, it can be speculated that there is a link between the association of huntingtin/Sh3d2c and the pathogenesis of Huntington disease.