ZIP Proteins Research Articles

Zinc and Its Impact on the Function of the Testicle and Epididymis.

Zinc (Zn) is an essential trace element; it exhibits a plethora of physiological properties and biochemical functions. It plays a pivotal role in regulating the cell cycle, apoptosis, and DNA organization, as well as in protein, lipid, and carbohydrate metabolism. Among other important processes, Zn plays an essential role in reproductive health. The ZIP and ZnT proteins are responsible for the mobilization of Zn within the cell. Zn is an inert antioxidant through its interaction with a variety of proteins and enzymes to regulate the redox system, including metallothioneins (MTs), metalloenzymes, and gene regulatory proteins. The role of Zn in the reproductive system is of great importance; processes, such as spermatogenesis and sperm maturation that occur in the testicle and epididymis, respectively, depend on this element for their development and function. Zn modulates the synthesis of androgens, such as testosterone, for these reproductive processes, so Zn deficiency is related to alterations in sperm parameters that lead to male infertility.

An acidic loop in the forkhead-associated domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at double-strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover-specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a 5-amino acid sequence, RPSKR, located between the DNA-binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full-length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a noncanonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt 2-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint and, in certain circumstances, exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

An acidic loop in the FHA domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at the double strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present. Mek1 interacts with Ndt80 through a five amino acid sequence, RPSKR, located between the DNA binding and activation domains of Ndt80. AlphaFold Multimer modeling of a fragment of Ndt80 containing the RPSKR motif and full length Mek1 indicated that RPSKR binds to an acidic loop located in the Mek1 FHA domain, a non-canonical interaction with this motif. A second protein, the 5'-3' helicase Rrm3, similarly interacts with Mek1 through an RPAKR motif and is an in vitro substrate of Mek1. Genetic analysis using various mutants in the MEK1 acidic loop validated the AlphaFold model, in that they specifically disrupt two-hybrid interactions with Ndt80 and Rrm3. Phenotypic analyses further showed that the acidic loop mutants are defective in the meiotic recombination checkpoint, and in certain circumstances exhibit more severe phenotypes compared to the NDT80 mutant with the RPSKR sequence deleted, suggesting that additional, as yet unknown, substrates of Mek1 also bind to Mek1 using an RPXKR motif.

Roles of zinc in cancers: From altered metabolism to therapeutic applications.

Zinc (Zn) is a crucial trace element involved in various cellular processes, including oxidative stress, apoptosis and immune response, contributing to cellular homeostasis. Dysregulation of Zn homeostasis occurs in certain cancers. This review discusses the role of Zn in cancer and its associated components, such as Zn-related proteins, their potential as biomarkers and the use of Zn-based strategies for tumor treatment. ZIP and ZnT proteins regulate Zn metabolism under normal conditions, but their expression is aberrant in cancer. These Zn proteins can serve as prognostic or diagnostic biomarkers, aiding in early cancer detection and disease monitoring. Moreover, targeting Zn and its pathways offers potential therapeutic approaches for cancer treatment. Modulating Zn biodistribution within cells using metal-binding agents allows for the control of downstream signaling pathways. Direct utilization of zinc as a therapeutic agent, including Zn supplementation or Zn oxide nanoparticle administration, holds promise for improving the prognosis of cancer patients.

Single-molecule quantification of the oligomeric state of ZIP transporters in mammalian cells with fluorescence correlation spectroscopy.

The SLC39 family of transporters, otherwise known as ZIPs for Zrt and Irt-like Proteins, function to increase cytosolic levels of transition metals. ZIP transporters have been identified at all phylogenetic levels and are members of the SoLute Carrier (SLC) superfamily. There are fourteen ZIP transporters encoded in the human genome. ZIP transmembrane proteins are expressed in the plasma membrane or membranes of intracellular organelles and have unique expression profiles across cell types. While direct structural efforts including x-ray crystallography, NMR and ab initio approaches have been effective tools in elucidating the structure of ZIPs, direct elucidation of the oligomeric state of these proteins is essential in understanding how wild type ZIP proteins function and whether mutations alter the oligomeric state of ZIPs. Unfortunately, several tools to quantify oligomeric states of proteins require overexpression of proteins which can lead to artifacts in experimental results. In contrast, fluorescence correlation spectroscopy (FCS) is a single-molecule technique which can be used to quantify the oligomeric state of transmembrane proteins. FCS takes advantage of the observation that the molecular brightness of a cluster of fluorescent molecules is directly proportional to the number of fluorescent molecules within the protein complex. This chapter describes how to implement FCS, focused on ZIP transporters, to quantify the oligomeric state of transmembrane in vivo. Included within this chapter are procedures to design constructs for experiments, transfection of mammalian cells as well as data acquisition and analysis. Taken together, FCS is a powerful mechanism to investigate the oligomeric state of proteins embedded within membranes of cells.

Considerations in production of the prokaryotic ZIP family transporters for structural and functional studies.

Zinc ions play essential roles as components of enzymes and many other important biomolecules, and are associated with numerous diseases. The uptake of Zn2+ and other metal ions require a widely distributed transporter protein family called Zrt/Irt-like Proteins (ZIP family), the majority members of which tend to have eight transmembrane helices with both N- and C- termini located on the extracellular or periplasmic side. Their small sizes and dynamic conformations bring many difficulties in their production for structural studies either by crystallography or Cryo-EM. Here, we summarize the problems that may encounter at the various steps of processing the ZIP proteins from gene to structural and functional studies, and provide some solutions and examples from our and other labs for the cloning, expression, purification, stability screening, metal ion transport assays and structural studies of prokaryotic ZIP family transporters using Escherichia coli as a heterologous host.

Histone variant H2A.Z promotes meiotic chromosome axis organization in Saccharomyces cerevisiae.

Progression through meiosis is associated with significant reorganization of chromosome structure, regulated in part by changes in histones and chromatin. Prior studies observed defects in meiotic progression in yeast strains lacking the linker histone H1 or variant histone H2A.Z. To further define the contributions of these chromatin factors, we have conducted genetic and cytological analysis of cells undergoing meiosis in the absence of H1 and H2A.Z. We find that a spore viability defect observed in strains lacking H2A.Z can be partially suppressed if cells also lack histone H1, while the combined loss of both H1 and H2A.Z is associated with elevated gene conversion events. Cytological analysis of Red1 and Rec8 staining patterns indicates that a subset of cells lacking H2A.Z fail to assemble a proper chromosome axis, and the staining pattern of the synaptonemal complex protein Zip1 in htz1Δ/htz1Δ cells mimics that of cells deficient for Rec8-dependent meiotic cohesion. Our results suggest a role for H2A.Z in the establishment or maintenance of the meiotic chromosome axis, possibly by promoting the efficient chromosome cohesion.

Genomic characterization of ZIP genes in pigeonpea (CcZIP) and their expression analysis among the genotypes with contrasting host response to pod borer.

The online version contains supplementary material available at 10.1007/s12298-021-01111-1.

Redundant roles of four ZIP family members in zinc homeostasis and seed development in Arabidopsis thaliana.

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana.

DNA Helicase Mph1FANCM Ensures Meiotic Recombination between Parental Chromosomes by Dissociating Precocious Displacement Loops.

Meiotic pairing between parental chromosomes (homologs) is required for formation of haploid gametes. Homolog pairing depends on recombination initiation via programmed double-strand breaks (DSBs). Although DSBs appear prior to pairing, the homolog, rather than the sister chromatid, is used as repair partner for crossing over. Here, we show that Mph1, the budding yeast ortholog of Fanconi anemia helicase FANCM, prevents precocious DSB strand exchange between sister chromatids before homologs have completed pairing. By dissociating precocious DNA displacement loops (D-loops) between sister chromatids, Mph1FANCM ensures high levels of crossovers and non-crossovers between homologs. Later-occurring recombination events are protected from Mph1-mediated dissociation by synapsis protein Zip1. Increased intersister repair in absence of Mph1 triggers a shift among remaining interhomolog events from non-crossovers to crossover-specific strand exchange, explaining Mph1's apparent anti-crossover function. Our findings identify temporal coordination between DSB strand exchange and homolog pairing as a critical determinant for recombination outcome.

Metal transporter encoding gene families in Fabaceae: III. The zinc-iron permease (ZIP) gene family

In plants, Zinc and Iron are transported through the membrane by proteins belonging to Zinc-Iron permease (ZIP: ZRT/IRT-like Protein). In this work, the ZIP gene families were identified in the genome of five legume species. The results demonstrated that the ZIPs were belonged to a multigeneic family in each species including soybean (28 genes), Medicago truncalata (16 genes), chickpea (7 genes), pigeon pea (12 genes), and Lotus japonicus (15 genes). Each gene contained from one to twelve introns. ZIP proteins possessed a conserved histidine-rich motif. Most of these proteins contained eight putative transmembrane domains and were predicted to be localized in plasma membranes. The phylogeny analysis showed that the legume ZIPs were classified into four main groups, each of which includes many subgroups. The group I contained the ZIP members of five examined plants. Moreover, the phylogeny showed gene gain events (expansion) in group I and gene loss events in other groups. The gene expansion in group I is likely to have arisen mainly from recent duplication events of ZIP genes in the examined legume plants, after specialization. The expression analysis showed that all of ZIP genes were expressed in all of the examined tissues in L. japonicus. The expression level of ZIP members was not similar in different tissues of the plant. Some ZIP genes were predominantly expressed in certain tissues for most of the legume species investigated.

Modulation of Symbiotic Compatibility by Rhizobial Zinc Starvation Machinery.

Pathogenic bacteria need high-affinity zinc uptake systems to counteract the nutritional immunity exerted by infected hosts. However, our understanding of zinc homeostasis in mutualistic systems such as the rhizobium-legume symbiosis is limited. Here, we show that the conserved high-affinity zinc transporter ZnuABC and accessory transporter proteins (Zip1, Zip2, and c06450) made cumulative contributions to nodulation of the broad-host-range strain Sinorhizobium fredii CCBAU45436. Zur acted as a zinc-dependent repressor for the znuC-znuB-zur operon, znuA, and c06450 by binding to the associated Zur box, but did not regulate zip1 and zip2 ZnuABC was the major zinc transporter. Combined mutants lacking znuA and one of the three accessory genes had more severe defects in nodulation and growth under zinc starvation conditions than the znuA mutant, though rhizoplane colonization by these mutants was not impaired. In contrast to the elite strain CCBAU45436, more drastic symbiotic defects were observed for the znuA mutants of other Sinorhizobium strains, which lack at least one of the three accessory genes in their genomes and are characterized by their limited host range and geographical distribution. The znu-derived mutants showed a higher expression level of nod genes involved in Nod factor biosynthesis and a reduced expression of genes encoding a type three secretion system and its effector NopP, which can interfere with the host immune system. Application of exogenous zinc restored the nodulation ability of these znu-derived mutants. Therefore, the conserved ZnuABC and accessory components in the zinc starvation machinery play an important role in modulating symbiotic compatibility.IMPORTANCE The rhizobium-legume symbiosis contributes around 65% of biological nitrogen fixation in agriculture systems and is critical for sustainable agriculture by reducing the amount of chemical nitrogen fertilizer being used. Rhizobial inocula have been commercialized for more than 100 years, but the efficiency of inoculation can vary among legume cultivars, field sites, and years. These long-lasting challenging problems impede the establishment of a sustainable agriculture, particularly in developing countries. Here, we report that rhizobial zinc starvation machinery containing a conserved high-affinity zinc transporter and accessory components makes cumulative contributions to modulating rhizobial symbiotic compatibility. This work highlights a critical role of largely unexplored nutritional immunity in the rhizobium-legume symbiosis, which makes zinc starvation machinery an attractive target for improving rhizobial symbiotic compatibility.

Distinct Functions in Regulation of Meiotic Crossovers for DNA Damage Response Clamp Loader Rad24(Rad17) and Mec1(ATR) Kinase.

The number and distribution of meiotic crossovers (COs) are highly regulated, reflecting the requirement for COs during the first round of meiotic chromosome segregation. CO control includes CO assurance and CO interference, which promote at least one CO per chromosome bivalent and evenly-spaced COs, respectively. Previous studies revealed a role for the DNA damage response (DDR) clamp and the clamp loader in CO formation by promoting interfering COs and interhomolog recombination, and also by suppressing ectopic recombination. In this study, we use classical tetrad analysis of Saccharomyces cerevisiae to show that a mutant defective in RAD24, which encodes the DDR clamp loader (RAD17 in other organisms), displayed reduced CO frequencies on two shorter chromosomes (III and V), but not on a long chromosome (chromosome VII). The residual COs in the rad24 mutant do not show interference. In contrast to rad24, mutants defective in the ATR kinase homolog Mec1, including a mec1 null and a mec1 kinase-dead mutant, show slight or few defects in CO frequency. On the other hand, mec1 COs show defects in interference, similar to the rad24 mutant. Our results support a model in which the DDR clamp and clamp-loader proteins promote interfering COs by recruiting pro-CO Zip, Mer, and Msh proteins to recombination sites, while the Mec1 kinase regulates CO distribution by a distinct mechanism. Moreover, CO formation and its control are implemented in a chromosome-specific manner, which may reflect a role for chromosome size in regulation.

An extracellular histidine-containing motif in the zinc transporter ZIP4 plays a role in zinc sensing and zinc-induced endocytosis in mammalian cells

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and also plays a structural role in numerous proteins. Zinc transporter ZIP4 (ZIP4) is a zinc importer required for dietary zinc uptake in the intestine and other cell types. Studies in cultured cells have reported that zinc stimulates the endocytosis of plasma membrane-localized ZIP4 protein, resulting in reduced cellular zinc uptake. Thus, zinc-regulated trafficking of ZIP4 is a key means for regulating cellular zinc homeostasis, but the underlying mechanisms are not well understood. In this study, we used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Moreover, using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane. These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells.

The trafficking of metal ion transporters of the Zrt- and Irt-like protein family.

Metal ion transporters of the Zrt- and Irt-like protein (ZIP, or SLC39A) family transport zinc, iron, manganese and/or cadmium across cellular membranes and into the cytosol. The 14 human ZIP family proteins are expressed in a wide variety of tissues and function in many different cellular processes. Many of these proteins (including ZIP1, 2, 3, 4, 5, 6/10, 8, 9, 11, 12, 14) are situated, at least some of the time, on the plasma membrane, where they mediate metal ion uptake into cells. Their level on the cell surface can be controlled rapidly via protein trafficking in response to the ions they transport. For example, the cell surface level of many ZIPs (including ZIP1, 3, 4, 8 and 12) is mediated by the available concentration of zinc. Zinc depletion causes a decrease in endocytosis and degradation, resulting in more ZIP on the surface to take up the essential ion. ZIP levels on the cell surface are a balance between endocytosis, recycling and degradation. We review the trafficking mechanisms of human ZIP proteins, highlighting possible targeting motifs and suggesting a model of zinc-mediated endocytic trafficking. We also provide two possible models for ZIP14 trafficking and degradation.

Elemental Profiling of Rice FOX Lines Leads to Characterization of a New Zn Plasma Membrane Transporter, OsZIP7.

Iron (Fe) and zinc (Zn) are essential micronutrients required for proper development in both humans and plants. Rice (Oryza sativa L.) grains are the staple food for nearly half of the world’s population, but a poor source of metals such as Fe and Zn. Populations that rely on milled cereals are especially prone to Fe and Zn deficiencies, the most prevalent nutritional deficiencies in humans. Biofortification is a cost-effective solution for improvement of the nutritional quality of crops. However, a better understanding of the mechanisms underlying grain accumulation of mineral nutrients is required before this approach can achieve its full potential. Characterization of gene function is more time-consuming in crops than in model species such as Arabidopsis thaliana. Aiming to more quickly characterize rice genes related to metal homeostasis, we applied the concept of high throughput elemental profiling (ionomics) to Arabidopsis lines heterologously expressing rice cDNAs driven by the 35S promoter, named FOX (Full Length Over-eXpressor) lines. We screened lines expressing candidate genes that could be used in the development of biofortified grain. Among the most promising candidates, we identified two lines ovexpressing the metal cation transporter OsZIP7. OsZIP7 expression in Arabidopsis resulted in a 25% increase in shoot Zn concentrations compared to non-transformed plants. We further characterized OsZIP7 and showed that it is localized to the plasma membrane and is able to complement Zn transport defective (but not Fe defective) yeast mutants. Interestingly, we showed that OsZIP7 does not transport Cd, which is commonly transported by ZIP proteins. Importantly, OsZIP7-expressing lines have increased Zn concentrations in their seeds. Our results indicate that OsZIP7 is a good candidate for developing Zn biofortified rice. Moreover, we showed the use of heterologous expression of genes from crops in A. thaliana as a fast method for characterization of crop genes related to the ionome and potentially useful in biofortification strategies.

Biphasic zinc compartmentalisation in a human fungal pathogen.

Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH-dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2Δ growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.

Reduced dosage of the chromosome axis factor Red1 selectively disrupts the meiotic recombination checkpoint in Saccharomyces cerevisiae.

Meiotic chromosomes assemble characteristic “axial element” structures that are essential for fertility and provide the chromosomal context for meiotic recombination, synapsis and checkpoint signaling. Whether these meiotic processes are equally dependent on axial element integrity has remained unclear. Here, we investigated this question in S. cerevisiae using the putative condensin allele ycs4S. We show that the severe axial element assembly defects of this allele are explained by a linked mutation in the promoter of the major axial element gene RED1 that reduces Red1 protein levels to 20–25% of wild type. Intriguingly, the Red1 levels of ycs4S mutants support meiotic processes linked to axis integrity, including DNA double-strand break formation and deposition of the synapsis protein Zip1, at levels that permit 70% gamete survival. By contrast, the ability to elicit a meiotic checkpoint arrest is completely eliminated. This selective loss of checkpoint function is supported by a RED1 dosage series and is associated with the loss of most of the cytologically detectable Red1 from the axial element. Our results indicate separable roles for Red1 in building the structural axis of meiotic chromosomes and mounting a sustained recombination checkpoint response.

Meiotic Centromere Coupling and Pairing Function by Two Separate Mechanisms in Saccharomyces cerevisiae.

In meiosis I, chromosomes become paired with their homologous partners and then are pulled toward opposite poles of the spindle. In the budding yeast, Saccharomyces cerevisiae, in early meiotic prophase, centromeres are observed to associate in pairs in a homology-independent manner; a process called centromere coupling. Later, as homologous chromosomes align, their centromeres associate in a process called centromere pairing. The synaptonemal complex protein Zip1 is necessary for both types of centromere association. We aimed to test the role of centromere coupling in modulating recombination at centromeres, and to test whether the two types of centromere associations depend upon the same sets of genes. The zip1-S75E mutation, which blocks centromere coupling but no other known functions of Zip1, was used to show that in the absence of centromere coupling, centromere-proximal recombination was unchanged. Further, this mutation did not diminish centromere pairing, demonstrating that these two processes have different genetic requirements. In addition, we tested other synaptonemal complex components, Ecm11 and Zip4, for their contributions to centromere pairing. ECM11 was dispensable for centromere pairing and segregation of achiasmate partner chromosomes; while ZIP4 was not required for centromere pairing during pachytene, but was required for proper segregation of achiasmate chromosomes. These findings help differentiate the two mechanisms that allow centromeres to interact in meiotic prophase, and illustrate that centromere pairing, which was previously shown to be necessary to ensure disjunction of achiasmate chromosomes, is not sufficient for ensuring their disjunction.

Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern.

During meiosis, chromosomes undergo a homology search in order to locate their homolog to form stable pairs and exchange genetic material. Early in prophase, chromosomes associate in mostly non-homologous pairs, tethered only at their centromeres. This phenomenon, conserved through higher eukaryotes, is termed centromere coupling in budding yeast. Both initiation of recombination and the presence of homologs are dispensable for centromere coupling (occurring in spo11 mutants and haploids induced to undergo meiosis) but the presence of the synaptonemal complex (SC) protein Zip1 is required. The nature and mechanism of coupling have yet to be elucidated. Here we present the first pairwise analysis of centromere coupling in an effort to uncover underlying rules that may exist within these non-homologous interactions. We designed a novel chromosome conformation capture (3C)-based assay to detect all possible interactions between non-homologous yeast centromeres during early meiosis. Using this variant of 3C-qPCR, we found a size-dependent interaction pattern, in which chromosomes assort preferentially with chromosomes of similar sizes, in haploid and diploid spo11 cells, but not in a coupling-defective mutant (spo11 zip1 haploid and diploid yeast). This pattern is also observed in wild-type diploids early in meiosis but disappears as meiosis progresses and homologous chromosomes pair. We found no evidence to support the notion that ancestral centromere homology plays a role in pattern establishment in S. cerevisiae post-genome duplication. Moreover, we found a role for the meiotic bouquet in establishing the size dependence of centromere coupling, as abolishing bouquet (using the bouquet-defective spo11 ndj1 mutant) reduces it. Coupling in spo11 ndj1 rather follows telomere clustering preferences. We propose that a chromosome size preference for centromere coupling helps establish efficient homolog recognition.