The 26S proteasome is a multisubunit complex responsible for degradation of ubiquitinated substrates, which plays a critical role in regulating various biological processes. To fully understand the function and regulation of the proteasome complex, an important step is to elucidate its subunit composition and posttranslational modifications. Toward this goal, a new affinity purification strategy has been developed using a derivative of the HB tag for rapid isolation of the human 26S proteasome complex for subsequent proteomic analysis. The purification of the complex is achieved from stable 293 cell lines expressing a HB-tagged proteasome subunit and by high-affinity streptavidin binding with TEV cleavage elution. The complete composition of the 26S proteasome complex, including recently assigned new subunits, is identified by LC-MS/MS. In addition, all known proteasome activator proteins and components involved in the ubiquitin-proteasome degradation pathway are identified. Aside from the subunit composition, the N-terminal modification and phosphorylation of the proteasome subunits have been characterized. Twelve novel phosphorylation sites from eight subunits have been identified, and N-terminal modifications are determined for 25 subunits, 12 of which have not been previously reported in mammals. We also observe different N-terminal processing of subunit Rpn2, which results in identification of two different N-termini of the protein. This work presents the first comprehensive characterization of the human 26S proteasome complex by affinity purification and tandem mass spectrometry. The detailed proteomic profiling obtained here is significant to future studies aiming at a complete understanding of the structure-function relationship of the human 26S proteasome complex.
A gene assigned to human chromosome 1q32-41 encodes a novel protein of 3,113 amino acids containing an internal tandem repeat of 177 amino acids. The protein, which we have named ''mitosin,'' was identified by direct binding to purified retinoblastoma protein in vitro with a region distantly related to the retinoblastoma protein-binding site of E2F-1. Mitosin is expressed throughout S, G 2 , and M phases of the cell cycle but is absent in G 1 . Its localization is dramatically reorganized from a rather homogeneous nuclear distribution in S phase to paired dots at the kinetochore/centromere region, to the spindle apparatus, and then to the midbody during M-phase progression. This spatial reorganization coincides closely with the temporal phosphorylation patterns of mitosin. Overexpression of N-terminally truncated mutants blocks cell cycle progression mainly at G 2 /M. These results suggest that mitosin may play an important role in mitotic-phase progression.
Active transport of proteins into the nucleus is mediated by interaction between the classical nuclear localization signals (NLSs) of the targeted proteins and the NLS receptor (importin) complex. This nuclear transport system is highly regulated and conserved in eukaryotes and is essential for cell survival. Using a fragment of BRCA1 containing the two NLS motifs as a bait for yeast two-hybrid screening, we have isolated four clones, one of which is importin ␣. Here we characterize one of the other clones identified, BRAP2, which is a novel gene and expressed as a 2-kilobase mRNA in human mammary epithelial cells and some but not all tissues of mice. The isolated full-length cDNA encodes a novel protein containing 600 amino acid residues with pI 6.04. Characteristic motifs of C2H2 zinc fingers and leucine heptad repeats are present in the middle and C-terminal regions of the protein, respectively. BRAP2 also shares significant homology with a hypothetical protein from yeast Saccharomyces cerevisiae, especially in the zinc finger region. Antibodies prepared against the C-terminal region of BRAP2 fused to glutathione S-transferase specifically recognize a cellular protein with a molecular size of 68 kDa, consistent with the size of the in vitro translated protein. Cellular BRAP2 is mainly cytoplasmic and binds to the NLS motifs of BRCA1 with similar specificity to that of importin ␣ in both two-hybrid assays in yeast and glutathione S-transferase pull-down assays in vitro. Other motifs such as the SV40 large T antigen NLS motif and the bipartite NLS motif found in mitosin are also recognized by BRAP2. Similarly, the yeast homolog of BRAP2 also binds to these NLS motifs in vitro. These results imply that BRAP2 may function as a cytoplasmic retention protein and play a role in regulating transport of nuclear proteins.The passage of macromolecules between the nucleus and the cytoplasm occurs through nuclear pores. Small macromolecules can diffuse through the nuclear pores at a rate inversely proportional to their mass. Proteins with molecular masses greater than 40 -60 kDa are actively transported through the nuclear pores. To be transported into the nucleus, the protein must either contain a nuclear localization signal or, if not, be bound to another protein that does (1, 2). This process requires at least four different factors acting in two distinct steps. The first step is mediated by importin ␣ (also termed karyopherin ␣) and importin  (also termed karyopherin ). The ␣ subunit is primarily responsible for NLS 1 recognition, whereas the  subunit appears to mediate docking to the nuclear pore complex. The second translocation step requires the small G protein Ran/TC4 and an interacting partner, p15 (3-13).The presence of a nuclear localization signal may not be sufficient to direct nuclear import. The target efficiency of NLS motifs can be modified by the presence of multiple NLS motifs within a protein, by modifications of the flanking sequences, and by the accessibility of the NLSs to the import machinery (1...
Mitochondrial dysfunction has been a hallmark of cancer. However, whether it plays a causive role awaits to be elucidated. Here, using an animal model derived from inactivation of SUV3, a mitochondrial helicase, we demonstrated that mSuv3+/− mice harbored increased mtDNA mutations and decreased mtDNA copy numbers, leading to tumor development in various sites and shortened lifespan. These phenotypes were transmitted maternally, indicating the etiological role of the mitochondria. Importantly, reduced SUV3 expression was observed in human breast tumor specimens compared to corresponding normal tissues in two independent cohorts. These results demonstrated for the first time that maintaining mtDNA integrity by SUV3 helicase is critical for cancer suppression.
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