Molecular epidemiology of human polyomavirus JC in the Biaka Pygmies and Bantu of Central Africa

The proteasome consists of a 20S proteolytic core particle (CP) and a 19S regulatory particle (RP), which selects ubiquitinated substrates for translocation into the CP. An eight-subunit subcomplex of the RP, the lid, can be dissociated from proteasomes prepared from a deletion mutant for Rpn10, an RP subunit. A second subcomplex, the base, contains all six proteasomal ATPases and links the RP to the CP. The base is sufficient to activate the CP for degradation of peptides or a nonubiquitinated protein, whereas the lid is required for ubiquitin-dependent degradation. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The lid subunits share sequence motifs with components of the COP9/signalosome complex and eIF3, suggesting that these functionally diverse particles have a common evolutionary ancestry.     

Evidence for the participation of the proteasome and calpain in early phases of muscle cell differentiation

Objectives were to investigate the role of the proteasome and m-calpain to muscle cell differentiation. Accordingly, we investigated the effects of lactacystin, a proteasome inhibitor, and calpain inhibitor-II (CI-II) on L8 muscle cell differentiation and assessed concentrations of proteasomal and calpain subunit mRNAs during differentiation. L8 myoblasts were induced to differentiate by culturing in mitogen-depleted medium. To assess the importance of the proteasome and calpain to differentiation, we examined effects of lactacystin and CI-II on creatine kinase (CK) activity. In the absence of inhibitor, CK activity was detectable within 48 h of mitogen depletion and myotubes were formed. Addition of lactacystin or CI-II to cultures drastically reduced CK activity and prevented formation of myotubes. Hence, proteasome and calpain are both necessary for differentiation. In order to identify which proteasomal subunits were regulated during differentiation, we examined the concentrations of two 20S core subunits (C8 and C9) and three 22S ATPases (MSS1, S4 and TBP1) during differentiation. Concentrations of m-calpain and beta-tubulin mRNAs were also assessed. Differentiation was associated with slight increases (ca. 30%) in concentrations of mRNAs encoding the proteasomal 20S core subunits (C8 and C9) and with large increases (approximately 2-fold) in mRNAs encoding the regulatory subunit ATPases. m-calpain mRNA concentration also increased two-fold following mitogen depletion. beta-Tubulin mRNA concentration remained unchanged early in the differentiation process and thereafter declined. Of interest, changes in proteasomal and m-calpain mRNAs occurred within 6-24 h of mitogen depletion (i.e., at least 24-36 h prior to detectable changes in creatine kinase activity). These results indicate that changes in expression of proteasome and calpains subunits occur early in the differentiation process. These changes may be required for the normal course of differentiation to proceed. Differentiation is associated with larger changes in proteasomal ATPase mRNAs than in 20S core particle mRNAs indicating that either turnover rates of the 22S ATPase subunits are more rapid in differentiating cells than of the 20S core particles or that functions of the regulatory subunits become more important during muscle cell differentiation.     

Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome

A family of ATPases resides within the regulatory particle of the proteasome. These proteins (Rpt1-Rpt6) have been proposed to mediate substrate unfolding, which may be required for translocation of substrates through the channel that leads from the regulatory particle into the proteolytic core particle. To analyze the role of ATP hydrolysis in protein breakdown at the level of the individual ATPase, we have introduced equivalent site-directed mutations into the ATPbinding motif of each RPT gene. Non-conservative substitutions of the active-site lysine were lethal in four of six cases, and conferred a strong growth defect in two cases. Thus, the ATPases are not functionally redundant, despite their multiplicity and sequence similarity. Degradation of a specific substrate can be inhibited by ATP-binding-site substitutions in many of the Rpt proteins, indicating that they co-operate in the degradation of individual substrates. The phenotypic defects of the different rpt mutants were strikingly varied. The most divergent phenotype was that of the rpt1 mutant, which was strongly growth defective despite showing no general defect in protein turnover. In addition, rpt1 was unique among the rpt mutants in displaying a G1 cell-cycle defect. Proteasomes purified from an rpt2 mutant showed a dramatic inhibition of peptidase activity, suggesting a defect in gating of the proteasome channel. In summary, ATP promotes protein breakdown by the proteasome through multiple mechanisms, as reflected by the diverse phenotypes of the rpt mutants.     

Platelet endothelial cell adhesion molecule-1 expression during mouse postimplantation development

The proteasome is an unusually large multisubunit proteolytic complex, consisting of a central catalytic machine (equivalent to the 20S proteasome) and two terminal regulatory subcomplexes, termed PA700 or PA28, that are attached to both ends of the central portion in opposite orientations to form the enzymatically active proteasome. Totally about 40 subunits with sizes of 20-110 kDa are assembled to form two types of the proteasomal complexes with the same catalytic core and different regulatory modules. To date, cDNAs or genes encoding almost all subunits of human and the budding yeast proteasomes have been isolated by molecular-biological techniques. In this minireview, I summarize briefly available information on the structure-function relationships of the proteasome acting as a protein death machinery.     

Progression of familial adenomatous polyposis (FAP) colonic cells after transfer of the src or polyoma middle T oncogenes: cooperation between src and HGF/Met in invasion

We have employed cDNA cloning to deduce the complete primary structures of p44.5 and p55, two subunits of PA700, a 700-kDa multisubunit regulatory complex of the human 26S proteasome. These polypeptides consist of 422 and 456 amino acids with calculated molecular masses of 47463 and 52903, and isoelectric points of 6.06 and 7.56, respectively. Computer-assisted homology analysis revealed high sequence similarities of p44.5 and p55 with yeast proteins whose functions are yet unknown. Disruption of the yeast genes, termed NAS4 and NAS5 (non-ATPase subunits 4 and 5), resulted in lethality, indicating that each of the two subunits is essential for proliferation of yeast cells.     

Proteasome assembly: biting the hand

Proteasome assembly is regulated to ensure the enzyme is inactive until its active sites are compartmentalized within an interior aqueous chamber. In yeast, this depends on a dedicated chaperone that is trapped within the nascent proteasome, and degraded on maturation of the proteolytic subunits.     

Molecular organization of the 20S proteasome gene family from Arabidopsis thaliana

The 20S proteasome is the proteolytic complex in eukaryotes responsible for degrading short-lived and abnormal intracellular proteins, especially those targeted by ubiquitin conjugation. The 700-kD complex exists as a hollow cylinder comprising four stacked rings with the catalytic sites located in the lumen. The two outer rings and the two inner rings are composed of seven different alpha and beta polypeptides, respectively, giving an alpha7/beta7/beta7/alpha7 symmetric organization. Here we describe the molecular organization of the 20S proteasome from the plant Arabidopsis thaliana. From an analysis of a collection of cDNA and genomic clones, we identified a superfamily of 23 genes encoding all 14 of the Arabidopsis proteasome subunits, designated PAA-PAG and PBA-PBG for Proteasome Alpha and Beta subunits A-G, respectively. Four of the subunits likely are encoded by single genes, and the remaining subunits are encoded by families of at least 2 genes. Expression of the alpha and beta subunit genes appears to be coordinately regulated. Three of the nine Arabidopsis proteasome subunit genes tested, PAC1 (alpha3), PAE1 (alpha5) and PBC2 (beta3), could functionally replace their yeast orthologs, providing the first evidence for cross-species complementation of 20S subunit genes. Taken together, these results demonstrate that the 20S proteasome is structurally and functionally conserved among eukaryotes and suggest that the subunit arrangement of the Arabidopsis 20S proteasome is similar if not identical to that recently determined for the yeast complex.     

Differential ubiquitin-dependent degradation of the yeast apo-cytochrome c isozymes

The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1- and iso-2-cytochrome c, which are encoded by the nuclear genes CYC1 and CYC7, respectively. The cytochromes c are synthesized in the cytosol, imported into mitochondria, and subsequently modified by the covalent attachment of heme through the action of cytochrome c heme lyase, which is encoded by CYC3. Apo-iso-2-cytochrome c but not apo-iso-1-cytochrome c was observed in cyc3(-) mutants. Furthermore, pulse-chase experiments previously demonstrated that the lack of apo-iso-1-cytochrome c was due to its rapid degradation. We report herein that this degradation of apo-iso-1-cytochrome c is dependent on ubiquitination and on the action of the proteasome. Diminished degradation of apo-iso-1-cytochrome c was observed in pre2-2 and pre1-1 mutants having altered proteasome subunits; in ubc1, ubc4, and ubc5 strains lacking one or more of the ubiquitin-conjugating enzymes; and in strains blocked in multi-ubiquitination by overproduction of the abnormal ubiquitin-K48R ubiquitin. In addition, we have used epitope-tagged ubiquitin to demonstrate that apo-iso-1-cytochrome c but not apo-iso-2-cytochrome c is ubiquitinated. Furthermore, the degradation of apo-iso-1-cytochrome c was diminished when the N-terminal region was replaced with the N-terminal region of apo-iso-2-cytochrome c, indicating that this region may be the target for degradation. We suggest that ubiquitin-dependent degradation of apo-iso-1-cytochrome c is part of the regulatory process controlling the preferential expression of the iso-cytochromes c.     

Subcellular distribution of proteasomes implicates a major location of protein degradation in the nuclear envelope-ER network in yeast

26S proteasomes are the key enzyme complexes responsible for selective turnover of short-lived and misfolded proteins. Based on the assumption that they are dispersed over the nucleoplasm and cytoplasm in all eukaryotic cells, we wanted to determine the subcellular distribution of 26S proteasomes in living yeast cells. For this purpose, we generated yeast strains that express functional green fluorescent protein (GFP) fusions of proteasomal subunits. An alpha subunit of the proteolytically active 20S core complex of the 26S proteasome, Pre6/YOL038w, as well as an ATPase-type subunit of the regulatory 19S cap complex, Cim5/YOL145w, were tagged with GFP. Both chimeras were shown to be incorporated completely into active 26S proteasomes. Microscopic analysis revealed that GFP-labelled 20S as well as 19S subunits are accumulated mainly in the nuclear envelope (NE)-endoplasmic reticulum (ER) network in yeast. These findings were supported by the co-localization and co-enrichment of 26S proteasomes with NE-ER marker proteins. A major location of proteasomal peptide cleavage activity was visualized in the NE-ER network, indicating that proteasomal degradation takes place mainly in this subcellular compartment in yeast.     

The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing

The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.     

NAD(+)-dependent isocitrate dehydrogenase from Arabidopsis thaliana. Characterization of two closely related subunits

Two cDNA clones which appear to encode different subunits of NAD(+)-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.41) were identified by homology searches from the Arabidopsis EST database. These cDNA clones were obtained and sequenced; both encoded full-length messages and displayed 82.7% nucleotide sequence identity over the coding region. The deduced amino acid sequences revealed preprotein lengths of 367 residues, with an amino acid identity of 86.1%. Genomic Southern blot analysis showed distinct single-copy genes for both IDH subunits. Both IDH subunits were expressed as recombinant proteins in Escherichia coli, and polyclonal antibodies were raised to each subunit. The Arabidopsis cDNA clones were expressed in Saccharomyces cerevisiae mutants which were deficient in either one or both of the yeast NAD(+)-dependent IDH subunits. The Arabidopsis cDNA clones failed to complement the yeast mutations; although both IDH-I and IDH-II were expressed at detectable levels, neither protein was imported into the mitochondria.     

Structural features of the human salivary mucin, MUC7

Specific mgi mutations in the alpha, beta or gamma subunits of the mitochondrial F1-ATPase have previously been found to suppress rho0 lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F1 is dependent on the F0 sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, rho0 mutants can be isolated, upon treatment with ethidium bromide, that lack six major F0 subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of rho0 mutants indicates that an F1-complex carrying a mgi mutation can assemble in the absence of F0 subunits and that suppression of rho0 lethality is an intrinsic property of the altered F1 particle.     

Identification, purification, and characterization of a PA700-dependent activator of the proteasome

The activity of the intracellular protease, the proteasome, is modulated by a number of specific regulatory proteins. One such regulator, PA700, is a 700,000-Da multisubunit protein that activates hydrolytic activities of the proteasome via a mechanism that involves the ATP-dependent formation of a proteasome-PA700 complex. Four subunits of PA700 have been shown previously to be members of a protein family that contains a consensus sequence for ATP binding, and purified PA700 expresses ATPase activity. We report here the identification, purification, and initial characterization of a new modulator of the proteasome. The modulator has no direct effect on the activity of the proteasome, but enhances PA700 activation of the proteasome by up to 8-fold. This activation is associated with the formation of a proteasome/PA700-containing complex that is significantly larger than that formed in its absence. The modulator has a native Mr of approximately 300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically distinct subunits with Mr values of 50,000, 42,000, and 27,000 (p50, p42, and p27, respectively). Amino acid sequence analysis of the subunits shows that p50 and p42 are members of the same ATP-binding protein family found in PA700. The p50 subunit is identical to TBP1, a protein previously reported to interact with human immunodeficiency virus Tat protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C. A. (1990) Science 248, 1650-1653), while the p42 subunit seems to be a new member of the family. The p27 subunit has no significant sequence similarity to any previously described protein. Both p50 and p42, but not p27, were also identified as components of PA700, increasing the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes that regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate proteasome activity.     

Adhesive interactions between medically important yeasts and bacteria

Yeasts are being increasingly identified as important organisms in human infections. Adhesive interactions between yeasts and bacteria may contribute to yeast retention at body sites. Methods for studying adhesive interactions between bacterial strains are well known, and range from simple macroscopic methods to flow chamber systems with complex image analysis capabilities. The adhesive interactions between bacteria and yeasts have been studied employing several of the methods originally developed for studying adhesive interactions between bacteria. However, in many of the methods employed the larger size of the yeasts as compared with bacteria results in strong sedimentation of the yeasts, often invalidating the method adapted. In addition, most methods are semi-quantitative and do not properly control mass transport. Consequently, adhesive interaction mechanisms between yeasts and bacteria identified hitherto, including lectin binding and protein-protein interactions, must be regarded with caution. Extensive physico-chemical characteristics of yeast cell surfaces are not available and a physico-chemical mechanism has not yet been put forth. A new method for quantifying adhesive interactions between yeasts and bacteria is proposed, based on the use of a parallel plate flow chamber, in which the influence of adhering bacteria upon the kinetics of yeast adhesion and aggregation of the adhering yeasts is quantitatively evaluated, under carefully controlled mass transport.     

A functional GTPase domain, but not its transmembrane domain, is required for function of the SRP receptor beta-subunit

The signal recognition particle and its receptor (SR) target nascent secretory proteins to the ER. SR is a heterodimeric ER membrane protein whose subunits, SRalpha and SRbeta, are both members of the GTPase superfamily. Here we characterize a 27-kD protein in Saccharomyces cerevisiae (encoded by SRP102) as a homologue of mammalian SRbeta. This notion is supported (a) by Srp102p's sequence similarity to SRbeta; (b) by its disposition as an ER membrane protein; (c) by its interaction with Srp101p, the yeast SRalpha homologue; and (d) by its role in SRP-dependent protein targeting in vivo. The GTP-binding site in Srp102p is surprisingly insensitive to single amino acid substitutions that inactivate other GTPases. Multiple mutations in the GTP-binding site, however, inactivate Srp102p. Loss of activity parallels a loss of affinity between Srp102p and Srp101p, indicating that the interaction between SR subunits is important for function. Deleting the transmembrane domain of Srp102p, the only known membrane anchor in SR, renders SR soluble in the cytosol, which unexpectedly does not significantly impair SR function. This result suggests that SR functions as a regulatory switch that needs to associate with the ER membrane only transiently through interactions with other components.     

In situ detection of tissue factor within the coronary intima in rat cardiac allograft vasculopathy

Proteasomes are multicatalytic complexes that function as the major proteolytic machinery in regulated protein degradation. The eukaryotic 20S proteasome proteolytic core structure comprises 14 different subunits: 7 alpha-type and 7 beta-type. DTS7 is a dominant temperature-sensitive (DTS) lethal mutation at 29 degrees that also acts as a recessive lethal at ambient temperatures. DTS7 maps to cytological position 71AB. Molecular characterization of DTS7 reveals that this is caused by a missense mutation in a beta-type subunit gene, beta2. A previously characterized DTS mutant, l(3)73Ai1, results from a missense mutation in another beta-type subunit gene, beta6. These two mutants share a very similar phenotype, show a strong allele-specific genetic interaction, and are rescued by the same extragenic suppressor, Su(DTS)-1. We propose that these mutants might act as "poison subunits," disrupting proteasome function in a dosage-dependent manner, and suggest how they may interact on the basis of the structure of the yeast 20S proteasome.