Browsing by Subject "yeast"

Now showing 1 - 6 of 6
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    Assembly of proteasome subunits into non-canonical complexes in vivo
    (Elsevier, 2017-01) Hammack, Lindsay J.; Kusmierczyk, Andrew R.; Department of Biology, School of Science
    Proteasomes exist in all domains of life. In general, they are comprised of a compartmentalized protease whose activity is modulated by one or more regulatory complexes with which it interacts. The quaternary structure of this compartmentalized protease, called the 20S proteasome, is absolutely conserved and consists of four heptameric rings stacked coaxially. The rings are made of structurally related α and β subunits. In eukaryotes, assembly factors chaperone the α and β subunits during 20S biogenesis. Here we demonstrate that proteasome subunits can assemble into structures other than the canonical 20S proteasome in vivo. Specifically, the yeast α4 subunit forms high molecular weight complexes whose abundance increases when proteasome function is compromised. Results from a disulfide crosslinking approach are consistent with these complexes being ring-shaped. Though several eukaryotic α subunits can form rings when expressed recombinantly in bacteria, this is the first evidence that such non-canonical complexes exist in vivo.
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    A Conserved 20S Proteasome Assembly Factor Requires a Cterminal HbYX Motif for Proteasomal Precursor Binding
    (2011-05) Kusmierczyk, Andrew R; Kunjappu, Mary J; Kim, Roger Y; Hochstrasser, Mark
    Dedicated chaperones facilitate the assembly of the eukaryotic proteasome, but how they function remains largely unknown. Here we show that a yeast 20S proteasome assembly factor, Pba1–Pba2, requires a previously overlooked C-terminal hydrophobic-tyrosine-X (HbYX) motif for function. HbYX motifs in proteasome activators open the 20S proteasome entry pore, but Pba1–Pba2 instead binds inactive proteasomal precursors. We discovered an archaeal ortholog of this factor, here named PbaA, that also binds preferentially to proteasomal precursors in a HbYX motif–dependent fashion using the same proteasomal α-ring surface pockets as are bound by activators. PbaA and the related PbaB protein can be induced to bind mature 20S proteasomes if the active sites in the central chamber are occupied by inhibitors. Our data are consistent with an allosteric mechanism in which the maturation of the proteasome active sites determines the binding of assembly chaperones, potentially shielding assembly intermediates or misassembled complexes from nonproductive associations until assembly is complete.
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    Erratum to: The loop-less tmCdc34 E2 mutant defective polyubiquitination in vitro and in vivo supports yeast growth in a manner dependent on Ubp14 and Cka2.
    (BMC, 2016) Lass, Agnieszka; Cocklin, Ross; Scaglione, Kenneth M.; Skowyra, Michael; Korolev, Sergey; Goebl, Mark; Skowyra, Dorota; Department of Biochemistry and Molecular Biology, IU School of Medicine
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    Integration of general amino acid control and TOR regulatory pathways in yeast
    (2010-05) Staschke, Kirk Alan; Wek, Ronald C.; Edenberg, Howard J.; Roach, Peter J.; Bard, Martin
    Two important nutrient sensing and regulatory pathways, the general amino acid control (GAAC) and the target of rapamycin (TOR), participate in the control of yeast growth and metabolism in response to changes in nutrient availability. Starvation for amino acids activates the GAAC through Gcn2p phosphorylation of the translation initiation factor eIF2 and preferential translation of GCN4, a transcription activator. TOR senses nitrogen availability and regulates transcription factors, such as Gln3p. We used microarray analyses to address the integration of the GAAC and TOR pathways in directing the yeast transcriptome during amino acid starvation and rapamycin treatment. We found that the GAAC is a major effector of the TOR pathway, with Gcn4p and Gln3p each inducing a similar number of genes during rapamycin treatment. While Gcn4p activates a common core of 57 genes, the GAAC directs significant variations in the transcriptome during different stresses. In addition to inducing amino acid biosynthetic genes, Gcn4p activates genes required for assimilation of secondary nitrogen sources, such as -amino-butyric acid (GABA). Gcn2p activation upon shifting to secondary nitrogen sources is suggested to occur by means of a dual mechanism. First, Gcn2p is induced by the release of TOR repression through a mechanism involving Sit4p protein phosphatase. Second, this eIF2 kinase is activated by select uncharged tRNAs, which were shown to accumulate during the shift to GABA medium. This study highlights the mechanisms by which the GAAC and TOR pathways are integrated to recognize changing nitrogen availability and direct the transcriptome for optimal growth adaptation.
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    A Screen for RNA-Binding Proteins in Yeast Indicates Dual Functions for Many Enzymes
    (2010-11) Scherrer, Tanja; Mittal, Nitish; Janga, Sarath Chandra; Gerber, André P.
    Hundreds of RNA-binding proteins (RBPs) control diverse aspects of post-transcriptional gene regulation. To identify novel and unconventional RBPs, we probed high-density protein microarrays with fluorescently labeled RNA and selected 200 proteins that reproducibly interacted with different types of RNA from budding yeast Saccharomyces cerevisiae. Surprisingly, more than half of these proteins represent previously known enzymes, many of them acting in metabolism, providing opportunities to directly connect intermediary metabolism with posttranscriptional gene regulation. We mapped the RNA targets for 13 proteins identified in this screen and found that they were associated with distinct groups of mRNAs, some of them coding for functionally related proteins. We also found that overexpression of the enzyme Map1 negatively affects the expression of experimentally defined mRNA targets. Our results suggest that many proteins may associate with mRNAs and possibly control their fates, providing dense connections between different layers of cellular regulation.
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    Some assembly required: dedicated chaperones in eukaryotic proteasome biogenesis
    (2008-09) Kusmierczyk, Andrew R; Hochstrasser, Mark
    The 26S proteasome is the key eukaryotic protease responsible for the degradation of intracellular proteins. Protein degradation by the 26S proteasome plays important roles in numerous cellular processes, including the cell cycle, differentiation, apoptosis, and the removal of damaged or misfolded proteins. How this 2.5-MDa complex, composed of at least 32 different polypeptides, is assembled in the first place is not well understood. However, it has become evident that this complicated task is facilitated by a framework of protein factors that chaperone the nascent proteasome through its various stages of assembly. We review here the known proteasome-specific assembly factors, most only recently discovered, and describe their potential roles in proteasome assembly, with an emphasis on the many remaining unanswered questions about this intricate process of assisted self-assembly.