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The Journal of Cell Biology Oct 2023Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical...
Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated ∼10-fold by phosphorylation and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than that of slow anchoring myosin-1s found on endosomal membranes. We, therefore, propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells.
Topics: Actins; Clathrin; Myosin Type I; Myosins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37549220
DOI: 10.1083/jcb.202303095 -
Nature Communications Dec 2023Acetylation of histones is a key post-translational modification that guides gene expression regulation. In yeast, the class I histone deacetylase containing Rpd3S...
Acetylation of histones is a key post-translational modification that guides gene expression regulation. In yeast, the class I histone deacetylase containing Rpd3S complex plays a critical role in the suppression of spurious transcription by removing histone acetylation from actively transcribed genes. The S. cerevisiae Rpd3S complex has five subunits (Rpd3, Sin3, Rco1, Eaf3, and Ume1) but its subunit stoichiometry and how the complex engages nucleosomes to achieve substrate specificity remains elusive. Here we report the cryo-EM structure of the complete Rpd3S complex bound to a nucleosome. Sin3 and two copies of subunits Rco1 and Eaf3 encircle the deacetylase subunit Rpd3 and coordinate the positioning of Ume1. The Rpd3S complex binds both trimethylated H3 tails at position lysine 36 and makes multiple additional contacts with the nucleosomal DNA and the H2A-H2B acidic patch. Direct regulation via the Sin3 subunit coordinates binding of the acetylated histone substrate to achieve substrate specificity.
Topics: Nucleosomes; Saccharomyces cerevisiae; Histones; Saccharomyces cerevisiae Proteins; Methylation; Acetylation; Acetyltransferases
PubMed: 38065958
DOI: 10.1038/s41467-023-43968-8 -
Science Advances Sep 2023H3K4 trimethylation (H3K4me3) is a conserved histone modification catalyzed by histone methyltransferase Set1, and its dysregulation is associated with pathologies....
H3K4 trimethylation (H3K4me3) is a conserved histone modification catalyzed by histone methyltransferase Set1, and its dysregulation is associated with pathologies. Here, we show that Set1 is intrinsically unstable and elucidate how its protein levels are controlled within cell cycle and during gene transcription. Specifically, Set1 contains a destruction box (D-box) that is recognized by E3 ligase APC/C and degraded by the ubiquitin-proteasome pathway. Cla4 phosphorylates serine 228 (S228) within Set1 D-box, which inhibits APC/C-mediated Set1 proteolysis. During gene transcription, PAF complex facilitates Cla4 to phosphorylate Set1-S228 and protect chromatin-bound Set1 from degradation. By modulating Set1 stability and its binding to chromatin, Cla4 and APC/C control H3K4me3 levels, which then regulate gene transcription, cell cycle progression, and chronological aging. In addition, there are 141 proteins containing the D-box that can be potentially phosphorylated by Cla4 to prevent their degradation by APC/C. We addressed the long-standing question about how Set1 stability is controlled and uncovered a new mechanism to regulate protein stability.
Topics: Cell Cycle; Cell Cycle Proteins; Chromatin; Histone Methyltransferases; Saccharomyces cerevisiae Proteins; Ubiquitin-Protein Ligases; Cdh1 Proteins
PubMed: 37774018
DOI: 10.1126/sciadv.adi7238 -
Biomolecules Dec 2023Deep neural network-based programs can be applied to protein structure modeling by inputting amino acid sequences. Here, we aimed to evaluate the AlphaFold2-modeled...
Deep neural network-based programs can be applied to protein structure modeling by inputting amino acid sequences. Here, we aimed to evaluate the AlphaFold2-modeled myocilin wild-type and variant protein structures and compare to the experimentally determined protein structures. Molecular dynamic and ligand binding properties of the experimentally determined and AlphaFold2-modeled protein structures were also analyzed. AlphaFold2-modeled myocilin variant protein structures showed high similarities in overall structure to the experimentally determined mutant protein structures, but the orientations and geometries of amino acid side chains were slightly different. The olfactomedin-like domain of the modeled missense variant protein structures showed fewer folding changes than the nonsense variant when compared to the predicted wild-type protein structure. Differences were also observed in molecular dynamics and ligand binding sites between the AlphaFold2-modeled and experimentally determined structures as well as between the wild-type and variant structures. In summary, the folding of the AlphaFold2-modeled MYOC variant protein structures could be similar to that determined by the experiments but with differences in amino acid side chain orientations and geometries. Careful comparisons with experimentally determined structures are needed before the applications of the in silico modeled variant protein structures.
Topics: Ligands; Eye Proteins; Cytoskeletal Proteins; Amino Acids; Glycoproteins
PubMed: 38275755
DOI: 10.3390/biom14010014 -
Nucleic Acids Research Dec 2023The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides....
The Msh2-Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2-Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2-Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2-Msh3 binding to 5' ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2-Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2-Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2-Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2-Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2-Msh3 can disrupt DNA replication and repair and highlights the role of Msh2-Msh3 protein abundance in Msh2-Msh3-mediated genomic instability.
Topics: Humans; DNA; DNA Mismatch Repair; DNA Repair; DNA-Binding Proteins; MutS Homolog 2 Protein; MutS Homolog 3 Protein; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Genomic Instability
PubMed: 37930834
DOI: 10.1093/nar/gkad934 -
PLoS Genetics Aug 2023The Tup1-Cyc8 complex in Saccharomyces cerevisiae was one of the first global co-repressors of gene transcription discovered. However, despite years of study, a full...
Systematic analysis of tup1 and cyc8 mutants reveals distinct roles for TUP1 and CYC8 and offers new insight into the regulation of gene transcription by the yeast Tup1-Cyc8 complex.
The Tup1-Cyc8 complex in Saccharomyces cerevisiae was one of the first global co-repressors of gene transcription discovered. However, despite years of study, a full understanding of the contribution of Tup1p and Cyc8p to complex function is lacking. We examined TUP1 and CYC8 single and double deletion mutants and show that CYC8 represses more genes than TUP1, and that there are genes subject to (i) unique repression by TUP1 or CYC8, (ii) redundant repression by TUP1 and CYC8, and (iii) there are genes at which de-repression in a cyc8 mutant is dependent upon TUP1, and vice-versa. We also reveal that Tup1p and Cyc8p can make distinct contributions to commonly repressed genes most likely via specific interactions with different histone deacetylases. Furthermore, we show that Tup1p and Cyc8p can be found independently of each other to negatively regulate gene transcription and can persist at active genes to negatively regulate on-going transcription. Together, these data suggest that Tup1p and Cyc8p can associate with active and inactive genes to mediate distinct negative and positive regulatory roles when functioning within, and possibly out with the complex.
Topics: Saccharomyces cerevisiae; DNA-Binding Proteins; Repressor Proteins; Saccharomyces cerevisiae Proteins; Nuclear Proteins; Transcription, Genetic; Gene Expression Regulation, Fungal; Fungal Proteins
PubMed: 37566621
DOI: 10.1371/journal.pgen.1010876 -
Protein Science : a Publication of the... Jun 2024While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial...
While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.
Topics: Histidine; Polyphosphates; Nitrilotriacetic Acid; Recombinant Proteins; Humans; Proteins
PubMed: 38747394
DOI: 10.1002/pro.5021 -
Nature Communications Oct 2023Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on...
Homologous recombination (HR) is an essential double-stranded DNA break repair pathway. In HR, Rad52 facilitates the formation of Rad51 nucleoprotein filaments on RPA-coated ssDNA. Here, we decipher how Rad52 functions using single-particle cryo-electron microscopy and biophysical approaches. We report that Rad52 is a homodecameric ring and each subunit possesses an ordered N-terminal and disordered C-terminal half. An intrinsic structural asymmetry is observed where a few of the C-terminal halves interact with the ordered ring. We describe two conserved charged patches in the C-terminal half that harbor Rad51 and RPA interacting motifs. Interactions between these patches regulate ssDNA binding. Surprisingly, Rad51 interacts with Rad52 at two different bindings sites: one within the positive patch in the disordered C-terminus and the other in the ordered ring. We propose that these features drive Rad51 nucleation onto a single position on the DNA to promote formation of uniform pre-synaptic Rad51 filaments in HR.
Topics: Cryoelectron Microscopy; DNA Repair; DNA, Single-Stranded; Protein Binding; Rad51 Recombinase; Rad52 DNA Repair and Recombination Protein; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 37798272
DOI: 10.1038/s41467-023-41993-1 -
Nucleic Acids Research Nov 2023Parental histone recycling is vital for maintaining chromatin-based epigenetic information during replication, yet its underlying mechanisms remain unclear. Here, we...
Parental histone recycling is vital for maintaining chromatin-based epigenetic information during replication, yet its underlying mechanisms remain unclear. Here, we uncover an unexpected role of histone chaperone FACT and its N-terminus of the Spt16 subunit during parental histone recycling and transfer in budding yeast. Depletion of Spt16 and mutations at its middle domain that impair histone binding compromise parental histone recycling on both the leading and lagging strands of DNA replication forks. Intriguingly, deletion of the Spt16-N domain impairs parental histone recycling, with a more pronounced defect observed on the lagging strand. Mechanistically, the Spt16-N domain interacts with the replicative helicase MCM2-7 and facilitates the formation of a ternary complex involving FACT, histone H3/H4 and Mcm2 histone binding domain, critical for the recycling and transfer of parental histones to lagging strands. Lack of the Spt16-N domain weakens the FACT-MCM interaction and reduces parental histone recycling. We propose that the Spt16-N domain acts as a protein-protein interaction module, enabling FACT to function as a shuttle chaperone in collaboration with Mcm2 and potentially other replisome components for efficient local parental histone recycling and inheritance.
Topics: Chromatin; DNA Helicases; Histone Chaperones; Histones; Molecular Chaperones; Nucleosomes; Saccharomyces cerevisiae Proteins; Transcriptional Elongation Factors; Multiprotein Complexes
PubMed: 37850662
DOI: 10.1093/nar/gkad846 -
Nature Communications Sep 2023The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe...
The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe peroxisome associated developmental disorders. Despite its pathophysiological importance, mechanistic details of the heterohexamer are not yet available. Here, we report cryoEM structures of Pex1/Pex6 from Saccharomyces cerevisiae, with an endogenous protein substrate trapped in the central pore of the catalytically active second ring (D2). Pairs of Pex1/Pex6(D2) subdomains engage the substrate via a staircase of pore-1 loops with distinct properties. The first ring (D1) is catalytically inactive but undergoes significant conformational changes resulting in alternate widening and narrowing of its pore. These events are fueled by ATP hydrolysis in the D2 ring and disengagement of a "twin-seam" Pex1/Pex6(D2) heterodimer from the staircase. Mechanical forces are propagated in a unique manner along Pex1/Pex6 interfaces that are not available in homo-oligomeric AAA-ATPases. Our structural analysis reveals the mechanisms of how Pex1 and Pex6 coordinate to achieve substrate translocation.
Topics: ATPases Associated with Diverse Cellular Activities; Cryoelectron Microscopy; Mutation; Peroxisomes; Proton-Translocating ATPases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 37741838
DOI: 10.1038/s41467-023-41640-9