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Cell Cycle (Georgetown, Tex.) Dec 2021In fission yeast, MBF-dependent transcription is required for cells to complete S phase. The MBF transcription factor is regulated through a complex feedback mechanism...
In fission yeast, MBF-dependent transcription is required for cells to complete S phase. The MBF transcription factor is regulated through a complex feedback mechanism that involves the co-repressors Yox1 and Nrm1 that are loaded onto MBF at the end of S phase, while positive transactivation is achieved through the constitutive binding of the co-activator Rep2. Here we show that Rep2 is required to fully recruit the chromatin remodelers SWI/SNF and RSC to MBF-regulated promoters. On the contrary, Nrm1 and Yox1, when bound to the MBF complex, block the approximation of these chromatin remodelers to MBF-regulated promoters. We propose that SWI/SNF and RSC are recruited to MBF-regulated genes, and RSC together with SAGA complex are important to regulate the G1-to-S transcriptional wave. Mutants of these remodeler complexes are highly sensitive when cells are exposed to insults that challenge DNA synthesis.
Topics: Cell Cycle Proteins; Chromatin; Homeodomain Proteins; Promoter Regions, Genetic; Repressor Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Trans-Activators; Transcription Factors
PubMed: 34843421
DOI: 10.1080/15384101.2021.2008203 -
Nature Structural & Molecular Biology Feb 2021Many proteins are transported into the endoplasmic reticulum by the universally conserved Sec61 channel. Post-translational transport requires two additional proteins,...
Many proteins are transported into the endoplasmic reticulum by the universally conserved Sec61 channel. Post-translational transport requires two additional proteins, Sec62 and Sec63, but their functions are poorly defined. In the present study, we determined cryo-electron microscopy (cryo-EM) structures of several variants of Sec61-Sec62-Sec63 complexes from Saccharomyces cerevisiae and Thermomyces lanuginosus and show that Sec62 and Sec63 induce opening of the Sec61 channel. Without Sec62, the translocation pore of Sec61 remains closed by the plug domain, rendering the channel inactive. We further show that the lateral gate of Sec61 must first be partially opened by interactions between Sec61 and Sec63 in cytosolic and luminal domains, a simultaneous disruption of which completely closes the channel. The structures and molecular dynamics simulations suggest that Sec62 may also prevent lipids from invading the channel through the open lateral gate. Our study shows how Sec63 and Sec62 work together in a hierarchical manner to activate Sec61 for post-translational protein translocation.
Topics: Endoplasmic Reticulum; Eurotiales; Heat-Shock Proteins; Membrane Transport Proteins; Models, Molecular; Protein Processing, Post-Translational; Protein Transport; SEC Translocation Channels; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 33398175
DOI: 10.1038/s41594-020-00541-x -
Molecular Biology of the Cell Apr 2018Cdc48/p97 and the ubiquilin family of UBA-UBL proteins are known for their role in the retrotranslocation of damaged proteins from the endoplasmic reticulum. We...
Cdc48/p97 and the ubiquilin family of UBA-UBL proteins are known for their role in the retrotranslocation of damaged proteins from the endoplasmic reticulum. We demonstrate that Cdc48 and the ubiquilin-like proteins in yeast also play a role in the anterograde trafficking of proteins, in this case the vacuolar protease, Cps1.
Topics: Autophagy; Carboxypeptidases; Cell Cycle Proteins; Endoplasmic Reticulum; Multivesicular Bodies; Nuclear Proteins; Nucleocytoplasmic Transport Proteins; Proteasome Endopeptidase Complex; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Valosin Containing Protein; Vesicular Transport Proteins
PubMed: 29444958
DOI: 10.1091/mbc.E17-11-0652 -
Proceedings of the National Academy of... Apr 2021The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an...
The Gcn pathway is conserved in all eukaryotes, including mammals such as humans, where it is a crucial part of the integrated stress response (ISR). Gcn1 serves as an essential effector protein for the kinase Gcn2, which in turn is activated by stalled ribosomes, leading to phosphorylation of eIF2 and a subsequent global repression of translation. The fine-tuning of this adaptive response is performed by the Rbg2/Gir2 complex, a negative regulator of Gcn2. Despite the wealth of available biochemical data, information on structures of Gcn proteins on the ribosome has remained elusive. Here we present a cryo-electron microscopy structure of the yeast Gcn1 protein in complex with stalled and colliding 80S ribosomes. Gcn1 interacts with both 80S ribosomes within the disome, such that the Gcn1 HEAT repeats span from the P-stalk region on the colliding ribosome to the P-stalk and the A-site region of the lead ribosome. The lead ribosome is stalled in a nonrotated state with peptidyl-tRNA in the A-site, uncharged tRNA in the P-site, eIF5A in the E-site, and Rbg2/Gir2 in the A-site factor binding region. By contrast, the colliding ribosome adopts a rotated state with peptidyl-tRNA in a hybrid A/P-site, uncharged-tRNA in the P/E-site, and Mbf1 bound adjacent to the mRNA entry channel on the 40S subunit. Collectively, our findings reveal the interaction mode of the Gcn2-activating protein Gcn1 with colliding ribosomes and provide insight into the regulation of Gcn2 activation. The binding of Gcn1 to a disome has important implications not only for the Gcn2-activated ISR, but also for the general ribosome-associated quality control pathways.
Topics: Binding Sites; Carrier Proteins; Molecular Dynamics Simulation; Peptide Elongation Factors; Protein Binding; Protein Serine-Threonine Kinases; RNA, Transfer, Amino Acyl; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Stress, Physiological
PubMed: 33790014
DOI: 10.1073/pnas.2022756118 -
PLoS Biology Jul 2019Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components...
Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components of the Atg1 kinase complex. Atg17 recruits and tethers Atg9-donor vesicles, and the corresponding Atg1 kinase complex induces the formation of nonselective autophagosomes. Atg11 initiates selective autophagy and coordinates the switch to nonselective autophagy by recruiting Atg17. The molecular function of Atg11 remained, however, less well understood. Here, we demonstrate that Atg11 is activated by cargo through a direct interaction with autophagy receptors. Activated Atg11 dimerizes and tethers Atg9 vesicles, which leads to the nucleation of phagophores in direct vicinity of cargo. Starvation reciprocally regulates the activity of both tethering factors by initiating the degradation of Atg11 while Atg17 is activated. This allows Atg17 to sequester and tether Atg9 vesicles independent of cargo to nucleate nonselective phagophores. Our data reveal insights into the molecular mechanisms governing cargo selection and specificity in autophagy.
Topics: Autophagosomes; Autophagy; Autophagy-Related Proteins; Binding, Competitive; Membrane Proteins; Protein Binding; Protein Multimerization; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Unilamellar Liposomes; Vesicular Transport Proteins
PubMed: 31356628
DOI: 10.1371/journal.pbio.3000377 -
Cell Reports Nov 2019Temporal control over protein phosphorylation and dephosphorylation is crucial for accurate chromosome segregation and for completion of the cell division cycle during...
Temporal control over protein phosphorylation and dephosphorylation is crucial for accurate chromosome segregation and for completion of the cell division cycle during exit from mitosis. In budding yeast, the Cdc14 phosphatase is thought to be a major regulator at this time, while in higher eukaryotes PP2A phosphatases take a dominant role. Here, we use time-resolved phosphoproteome analysis in budding yeast to evaluate the respective contributions of Cdc14, PP2A, and PP2A. This reveals an overlapping requirement for all three phosphatases during mitotic progression. Our time-resolved phosphoproteome resource reveals how Cdc14 instructs the sequential pattern of phosphorylation changes, in part through preferential recognition of serine-based cyclin-dependent kinase (Cdk) substrates. PP2A and PP2A in turn exhibit a broad substrate spectrum with some selectivity for phosphothreonines and a role for PP2A in sustaining Aurora kinase activity. These results illustrate synergy and coordination between phosphatases as they orchestrate phosphoproteome dynamics during mitotic progression.
Topics: Cell Cycle Proteins; Mitosis; Phosphoproteins; Protein Phosphatase 2; Protein Tyrosine Phosphatases; Proteome; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31722221
DOI: 10.1016/j.celrep.2019.10.041 -
Biochemistry Feb 2018The amyloid-based yeast prions are folded in-register parallel β-sheet polymers. Each prion can exist in a wide array of variants, with different biological properties...
The amyloid-based yeast prions are folded in-register parallel β-sheet polymers. Each prion can exist in a wide array of variants, with different biological properties resulting from different self-propagating amyloid conformations. Yeast has several anti-prion systems, acting in normal cells (without protein overexpression or deficiency). Some anti-prion proteins partially block prion formation (Ssb1,2p, ribosome-associated Hsp70s); others cure a large portion of prion variants that arise [Btn2p, Cur1p, Hsp104 (a disaggregase), Siw14p, and Upf1,2,3p, nonsense-mediated decay proteins], and others prevent prion-induced pathology (Sis1p, essential cytoplasmic Hsp40). Study of the anti-prion activity of Siw14p, a pyrophosphatase specific for 5-diphosphoinositol pentakisphosphate (5PP-IP5), led to the discovery that inositol polyphosphates, signal transduction molecules, are involved in [PSI+] prion propagation. Either inositol hexakisphosphate or 5PP-IP4 (or 5PP-IP5) can supply a function that is needed by nearly all [PSI+] variants. Because yeast prions are informative models for mammalian prion diseases and other amyloidoses, detailed examination of the anti-prion systems, some of which have close mammalian homologues, will be important for the development of therapeutic measures.
Topics: Amino Acid Transport Systems; Glutathione Peroxidase; HSP40 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Heat-Shock Proteins; Inositol; Molecular Chaperones; Nonsense Mediated mRNA Decay; Polyphosphates; Prions; Protein Tyrosine Phosphatases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 29377675
DOI: 10.1021/acs.biochem.7b01285 -
Nature Communications Nov 2019A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi. However, the dynamic regulations of polarisome...
A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi. However, the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown. We identify a previously functionally unknown polarisome member, actin-interacting-protein 5 (Aip5), which promotes actin assembly synergistically with formin Bni1. Aip5-C terminus is responsible for its activities by interacting with G-actin and Bni1. Through N-terminal intrinsically disordered region, Aip5 forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions. The molecular dynamics and reversibility of Aip5 condensates are regulated by scaffolding protein Spa2 via liquid-liquid phase separation both in vitro and in vivo. In the absence of Spa2, Aip5 condensates hamper cell growth and actin cable structures under stress treatment. The present study reveals the mechanisms of actin assembly for polarity establishment and the adaptation in stress conditions to protect actin assembly by protein phase separation.
Topics: Actins; Cell Enlargement; Cell Polarity; Crystallography, X-Ray; Cytoskeletal Proteins; Microfilament Proteins; Polymerization; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 31699995
DOI: 10.1038/s41467-019-13125-1 -
PloS One 2019Eukaryotic organelles share different components and establish physical contacts to communicate throughout the cell. One of the best-recognized examples of such...
Eukaryotic organelles share different components and establish physical contacts to communicate throughout the cell. One of the best-recognized examples of such interplay is the metabolic cooperation and crosstalk between mitochondria and peroxisomes, both organelles being functionally and physically connected and linked to the endoplasmic reticulum (ER). In Saccharomyces cerevisiae, mitochondria are linked to the ER by the ERMES complex that facilitates inter-organelle calcium and phospholipid exchanges. Recently, peroxisome-mitochondria contact sites (PerMit) have been reported and among Permit tethers, one component of the ERMES complex (Mdm34) was shown to interact with the peroxin Pex11, suggesting that the ERMES complex or part of it may be involved in two membrane contact sites (ER-mitochondria and peroxisome- mitochondria). This opens the possibility of exchanges between these three membrane compartments. Here, we investigated in details the role of each ERMES subunit on peroxisome abundance. First, we confirmed previous studies from other groups showing that absence of Mdm10 or Mdm12 leads to an increased number of mature peroxisomes. Secondly, we showed that this is not simply due to respiratory function defect, mitochondrial DNA (mtDNA) loss or mitochondrial network alteration. Finally, we present evidence that the contribution of ERMES subunits Mdm10 and Mdm12 to peroxisome number involves two different mechanisms.
Topics: Calcium; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Peroxisomes; Phospholipids; Point Mutation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 30908556
DOI: 10.1371/journal.pone.0214287 -
Proceedings of the National Academy of... Dec 2017Cells experience compressive stress while growing in limited space or migrating through narrow constrictions. To survive such stress, cells reprogram their intracellular...
Cells experience compressive stress while growing in limited space or migrating through narrow constrictions. To survive such stress, cells reprogram their intracellular organization to acquire appropriate mechanical properties. However, the mechanosensors and downstream signaling networks mediating these changes remain largely unknown. Here, we have established a microfluidic platform to specifically trigger compressive stress, and to quantitatively monitor single-cell responses of budding yeast in situ. We found that yeast senses compressive stress via the cell surface protein Mid2 and the calcium channel proteins Mid1 and Cch1, which then activate the Pkc1/Mpk1 MAP kinase pathway and calcium signaling, respectively. Genetic analysis revealed that these pathways work in parallel to mediate cell survival. Mid2 contains a short intracellular tail and a serine-threonine-rich extracellular domain with spring-like properties, and both domains are required for mechanosignaling. Mid2-dependent spatial activation of the Pkc1/Mpk1 pathway depolarizes the actin cytoskeleton in budding or shmooing cells, thereby antagonizing polarized growth to protect cells under compressive stress conditions. Together, these results identify a conserved signaling network responding to compressive mechanical stress, which, in higher eukaryotes, may ensure cell survival in confined environments.
Topics: Actin Cytoskeleton; Calcineurin; Calcium Channels; Calcium Signaling; Cell Survival; Cell Wall; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Mechanotransduction, Cellular; Membrane Glycoproteins; Microfluidics; Mitogen-Activated Protein Kinases; Protein Kinase C; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Stress, Mechanical
PubMed: 29196524
DOI: 10.1073/pnas.1709079114