Biology subjects

Voelkel-Meiman, K.

Publications and source records attributed to Voelkel-Meiman, K..

5 recordsLinked to original sources

bioRxiv2025

Crossover formation and coordinated assembly of synaptonemal complex relies on a direct interaction between Zip1 and Zip3

Several proteins collaborate to promote the crossover recombination events critical for accurate chromosome segregation during meiosis. How these "ZMM" factors (Zip2, Zip3, Zip4, Spo16, Mer3 and MutS{gamma}) collaboratively function remains incompletely understood. We previously reported that Zip3s abundance and activity rely on the synaptonemal complex (SC) component Zip1, and specifically on Zip1s N-terminal residues associated with crossovers and coupling SC assembly to the crossover pathway. Here, we demonstrate that Zip3 co-immunoprecipitates Zip1 from meiotic cells independent of recombination initiation and other ZMMs, and that Zip3s interaction with Zip1 relies on Zip1s N terminal residues. Co-expression and pull-down experiments in bacterial cells demonstrate that Zip1 and Zip3 interact directly. Experiments to identify Zip3 regions required for the Zip1 interaction unexpectedly revealed an incorrectly annotated translational start; we also determined that Zip3s N-terminal structured region is necessary and sufficient for the interaction, and a predicted coil downstream of Zip3s RING domain is essential for specific activities attributed to Zip1s N-terminal tip such as proximity labeling of Zip3 by Zip2 and the coupling of crossover recombination to SC assembly. Finally, we discovered that interaction with Zip1 protects Zip3 not only from proteasome-mediated degradation but also from post-translational modification when another ZMM is absent. We propose that direct interaction with Zip1s N terminus orients Zip3 within a nascent ZMM ensemble in a manner that facilitates crossover formation and the coupling of crossover intermediates to SC assembly, and furthermore ensures Zip3 remains both abundant and unmodified until all requisite ZMMs have joined the group.

genetics↗

bioRxiv2023

Proximity labeling reveals new insights into the relationships between meiotic recombination proteins in S. cerevisiae

Several protein ensembles facilitate MutS{gamma} crossover recombination and the associated process of synaptonemal complex (SC) assembly during meiosis, but the physical and functional relationships between the components involved remain obscure. We have employed proximity labeling as a phenotypic tool to discern functional relationships between meiotic recombination and SC proteins in S. cerevisiae, and to gain deeper insight into molecular deficits of crossover-defective mutants. We find that recombination initiation (Spo11) and the Mer3 helicase are dispensable for proximity labeling of the Zip3 E3 ligase by components of the ZZS ensemble (Zip2, Zip4 and Spo16) but are required for proximity labeling of Zip3 by Msh4, consistent with the possibility that MutS{gamma} joins Zip3 only after a specific recombination intermediate has been generated. Proximity labeling analysis of crossover-defective zip1 mutants suggests a key shared defect is a failure to assemble an early recombination ensemble where ZZS can properly engage Zip3. We furthermore discovered that Zip3s abundance within the meiotic cell is uniquely dependent on the presence of Zip1, and that the post-translational modification of Zip3 is promoted by most MutS{gamma} pathway proteins but countered by Zip1. Based on this and additional data, we propose a model whereby Zip1 stabilizes a functional, unmodified form of Zip3 until intermediate steps in recombination are complete. We also find that SC structural protein Ecm11 is proximity labeled by ZZS complex proteins in a Zip4-dependent manner, but by Zip3 and Msh4, at least in part, via a distinct pathway. Finally, streptavidin pulldowns followed by mass spectrometry on eleven different proximity labeling strains uncovers shared proximity targets of MutS{gamma}-associated proteins, some with known meiotic functions and others not yet implicated in a meiotic activity, highlighting the potential power of proximity labeling as a discovery tool.

genetics↗

bioRxiv2021

A Role for Synaptonemal Complex in Meiotic Mismatch Repair

During meiosis a large subset of interhomolog recombination repair intermediates form within the physical context of the synaptonemal complex (SC), a protein-rich structure assembled at the interface of aligned homologous chromosomes. However, the functional relationship between SC structure and homologous recombination remains poorly defined. In prior work we determined that tripartite SC is dispensable for recombination in S. cerevisiae; SC central element proteins Ecm11 and Gmc2 instead limit the number of recombination events. Here we report that while dispensable for recombination per se, SC central element proteins influence the processing of interhomolog recombination intermediates in a manner that minimizes errors in mismatch correction. Failure to correct mis-paired bases within heteroduplex at meiotic recombination sites leads to genotypically sectored colonies (post meiotic segregation events) arising from mitotic proliferation of mismatch-containing spores. We discovered an increase in post-meiotic segregation at the THR1 locus in cells lacking Ecm11 or Gmc2, or in the SC-deficient but crossover recombination-proficient zip1[{Delta}21-163] mutant. High-throughput sequencing of octad meiotic products revealed a genome-wide increase in recombination events with uncorrected mismatches in ecm11 mutants relative to wild type. Meiotic cells missing Ecm11 also display longer gene conversion tracts, but tract length alone does not account for the higher frequency of uncorrected mismatches. Interestingly, the per-nucleotide mismatch frequency is elevated in ecm11 mutants when analyzing all gene conversion tracts, but is similar between wild type and ecm11 if one considers only those events with uncorrected mismatches. Our data suggest that a subset of recombination events is similarly susceptible to mismatch repair errors in both wild type and ecm11 strains, but in ecm11 mutants many more recombination events fall into this inefficient repair category. Finally, we observe elevated post-meiotic segregation at THR1 in mutants with a dual deficiency in MutS{gamma}-mediated crossover recombination and SC assembly, but not in the mlh3 mutant, which lacks MutS{gamma} crossovers but has abundant SC. We propose that SC structure promotes efficient mismatch repair of joint molecule recombination intermediates resolved via both MutS{gamma}-associated and MutS{gamma}-independent pathways, and is the molecular basis for elevated post-meiotic segregation in both MutS{gamma} crossover-proficient (ecm11, gmc2) and MutS{gamma} crossover-deficient (msh4, zip3) strains.

genetics↗

bioRxiv2020

GCN sensitive protein translation in yeast

Levels of protein translation by ribosomes are governed both by features of the translation machinery as well as sequence properties of the mRNAs themselves. We focus here on a striking three-nucleotide periodicity, characterized by overrepresentation of GCN codons and underrepresentation of G at the second position of codons, that is observed in Open Reading Frames (ORFs) of mRNAs. Our examination of mRNA sequences in Saccharomyces cerevisiae revealed that this periodicity is particularly pronounced in the initial codons--the ramp region--of ORFs of genes with high protein expression. It is also found in mRNA sequences immediately following non-standard AUG start sites, located upstream or downstream of the standard annotated start sites of genes. To explore the possible influences of the ramp GCN periodicity on translation efficiency, we tested edited ramps with accentuated or depressed periodicity in two test genes, SKN7 and HMT1. Greater conformance to (GCN)n was found to significantly depress translation, whereas disrupting conformance had neutral or positive effects on translation. Our recent Molecular Dynamics analysis of a subsystem of translocating ribosomes in yeast revealed an interaction surface that H-bonds to the +1 codon that is about to enter the ribosome decoding center A site. The surface, comprised of 16S/18S rRNA C1054 and A1196 (E. coli numbering) and R146 of ribosomal protein Rps3, preferentially interacts with GCN codons, and we hypothesize that modulation of this mRNA-ribosome interaction may underlie GCN-mediated regulation of protein translation. Integration of our expression studies with large-scale reporter studies of ramp sequence variants suggests a model in which the C1054-A1196-R146 (CAR) interaction surface can act as both an accelerator and braking system for ribosome translation.

molecular biology↗

bioRxiv2018

Crossover recombination and synapsis are linked by adjacent regions within the N terminus of the Zip1 synaptonemal complex protein

Accurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes, which is often associated with the meiosis-specific MutS{gamma} complex. The recombination intermediates that give rise to MutS{gamma} interhomolog crossovers are embedded within a hallmark meiotic prophase structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known central region building blocks of the budding yeast SC, the Zip1 protein is unique for its SC-independent role in promoting MutS{gamma} crossovers. Here we report that adjacent regions within Zip1s unstructured N terminus encompass its crossover and SC assembly functions. We previously showed that deletion of Zip1 residues 21-163 abolishes tripartite SC assembly and prevents the robust SUMOylation of the SC central element component, Ecm11, but allows excess MutS{gamma} crossover recombination. We find the reciprocal phenotype when Zip1 residues 2-9 or 10-14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutS{gamma} crossovers are strongly diminished. Interestingly, Zip1 residues 2-9 or 2-14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutS{gamma} crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15-20 does not detectably prevent Zip3s localization at Zip1 polycomplex and supports some MutS{gamma} crossing over but prevents normal SC assembly and robust Ecm11 SUMOylation. These results highlight distinct N terminal regions that are differentially critical for Zip1s roles in crossover recombination and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors in close proximity to one another.

genetics↗