Interaction between VPS13A and the XK scramblase is important for VPS13A function in humans.

VPS13 family proteins form conduits between the membranes of different organelles through which lipids are transferred. In humans, there are four VPS13 paralogs, and mutations in the genes encoding each of them are associated with different inherited disorders. VPS13 proteins contain multiple conserved domains. The Vps13 adaptor-binding (VAB) domain binds to adaptor proteins that recruit VPS13 to specific membrane contact sites. This work demonstrates the importance of a different domain in VPS13A function. The pleckstrin homology (PH) domain at the C-terminal region of VPS13A is required to form a complex with the XK scramblase and for the co-localization of VPS13A with XK within the cell. Alphafold modeling was used to predict an interaction surface between VPS13A and XK. Mutations in this region disrupt both complex formation and co-localization of the two proteins. Mutant VPS13A alleles found in patients with VPS13A disease truncate the PH domain. The phenotypic similarities between VPS13A disease and McLeod syndrome caused by mutations in VPS13A and XK, respectively, argue that loss of the VPS13A-XK complex is the basis of both diseases.

Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane extension.

Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71-Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.

Genetic Dissection of Vps13 Regulation in Yeast Using Disease Mutations from Human Orthologs.

The VPS13 family of proteins have emerged as key players in intracellular lipid transport and human health. Humans have four different VPS13 orthologs, the dysfunction of which leads to different diseases. Yeast has a single VPS13 gene, which encodes a protein that localizes to multiple different membrane contact sites. The yeast vps13Δ mutant is pleiotropic, exhibiting defects in sporulation, protein trafficking, endoplasmic reticulum (ER)-phagy and mitochondrial function. Non-null alleles resulting from missense mutations can be useful reagents for understanding the multiple functions of a gene. The exceptionally large size of Vps13 makes the identification of key residues challenging. As a means to identify critical residues in yeast Vps13, amino acid substitution mutations from VPS13A, B, C and D, associated with human disease, were introduced at the cognate positions of yeast VPS13, some of which created separation-of-function alleles. Phenotypic analyses of these mutants have revealed that the promotion of ER-phagy is a fourth, genetically separable role of VPS13 and provide evidence that co-adaptors at the endosome mediate the activity of VPS13 in vacuolar sorting.

Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

During meiosis, programmed double strand breaks (DSBs) are repaired preferentially between homologs to generate crossovers that promote proper chromosome segregation at Meiosis I. In many organisms, there are two strand exchange proteins, Rad51 and the meiosis-specific Dmc1, required for interhomolog (IH) bias. This bias requires the presence, but not the strand exchange activity of Rad51, while Dmc1 is responsible for the bulk of meiotic recombination. How these activities are regulated is less well established. In dmc1Δ mutants, Rad51 is actively inhibited, thereby resulting in prophase arrest due to unrepaired DSBs triggering the meiotic recombination checkpoint. This inhibition is dependent upon the meiosis-specific kinase Mek1 and occurs through two different mechanisms that prevent complex formation with the Rad51 accessory factor Rad54: (i) phosphorylation of Rad54 by Mek1 and (ii) binding of Rad51 by the meiosis-specific protein Hed1. An open question has been why inhibition of Mek1 affects Hed1 repression of Rad51. This work shows that Hed1 is a direct substrate of Mek1. Phosphorylation of Hed1 at threonine 40 helps suppress Rad51 activity in dmc1Δ mutants by promoting Hed1 protein stability. Rad51-mediated recombination occurring in the absence of Hed1 phosphorylation results in a significant increase in non-exchange chromosomes despite wild-type levels of crossovers, confirming previous results indicating a defect in crossover assurance. We propose that Rad51 function in meiosis is regulated in part by the coordinated phosphorylation of Rad54 and Hed1 by Mek1.

Correction: Mek1 Down Regulates Rad51 Activity during Yeast Meiosis by Phosphorylation of Hed1.

[This corrects the article DOI: 10.1371/journal.pgen.1006226.].

In vitro reconstitution of the yeast spore wall dityrosine layer discloses the mechanism of its assembly.

In response to nutrient starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a dormant form of haploid cell termed a spore. The dityrosine layer forms the outermost layer of the wall of S. cerevisiae spores and endows them with resistance to environmental stresses. ll-Bisformyl dityrosine is the main constituent of the dityrosine layer, but the mechanism of its assembly remains elusive. Here, we found that ll-bisformyl dityrosine, but not ll-dityrosine, stably associated in vitro with dit1Δ spores, which lack the dityrosine layer. No other soluble cytosolic materials were required for this incorporation. In several aspects, the dityrosine incorporated in trans resembled the dityrosine layer. For example, dityrosine incorporation obscured access of the dye calcofluor white to the underlying chitosan layer, and ll-bisformyl dityrosine molecules bound to dit1Δ spores were partly isomerized to the dl-form. Mutational analyses revealed several spore wall components required for this binding. One was the chitosan layer located immediately below the dityrosine layer in the spore wall. However, ll-bisformyl dityrosine did not stably bind to chitosan particles, indicating that chitosan is not sufficient for this association. Several lines of evidence demonstrated that spore-resident proteins are involved in the incorporation, including the Lds proteins, which are localized to lipid droplets attached to the developing spore wall. In conclusion, our results provide insight into the mechanism of dityrosine layer formation, and the in vitro assay described here may be used to investigate additional mechanisms in spore wall assembly.

Congenital valvular defects associated with deleterious mutations in the PLD1 gene.

BACKGROUND: The underlying molecular aetiology of congenital heart defects is largely unknown. The aim of this study was to explore the genetic basis of non-syndromic severe congenital valve malformations in two unrelated families. METHODS: Whole-exome analysis was used to identify the mutations in five patients who suffered from severe valvular malformations involving the pulmonic, tricuspid and mitral valves. The significance of the findings was assessed by studying sporulation of yeast carrying a homologous Phospholipase D (PLD1) mutation, in situ hybridisation in chick embryo and echocardiography and histological examination of hearts of PLD1 knockout mice. RESULTS: Three mutations, p.His442Pro, p.Thr495fs32* and c.2882+2T>C, were identified in the PLD1 gene. The mutations affected highly conserved sites in the PLD1 protein and the p.His442Pro mutation produced a strong loss of function phenotype in yeast homologous mutant strain. Here we show that in chick embryos PLD1 expression is confined to the forming heart (E2-E8) and homogeneously expressed all over the heart during days E2-E3. Thereafter its expression decreases, remaining only adjacent to the atrioventricular valves and the right ventricular outflow tract. This pattern of expression follows the known dynamic patterning of apoptosis in the developing heart, consistent with the known role of PLD1 in the promotion of apoptosis. In hearts of PLD1 knockout mice, we detected marked tricuspid regurgitation, right atrial enlargement, and increased flow velocity, narrowing and thickened leaflets of the pulmonic valve. CONCLUSIONS: The findings support a role for PLD1 in normal heart valvulogenesis.

Post-transcriptional regulation in budding yeast meiosis.

The precise regulation of gene expression is essential for developmental processes in eukaryotic organisms. As an important post-transcriptional regulatory point, translational control is complementary to transcriptional regulation. Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental process controlled by a well-studied transcriptional cascade that drives the cell through the events of DNA replication, meiotic chromosome segregation, and spore assembly. Recent studies have revealed that as cells begin the meiotic divisions, translational regulation of gene expression fine tunes this transcriptional cascade. The significance and mechanisms of this translational regulation are beginning to emerge. These studies may also provide insights into translational regulation in germ cell development of multicellular organisms.

A highly redundant gene network controls assembly of the outer spore wall in S. cerevisiae.

The spore wall of Saccharomyces cerevisiae is a multilaminar extracellular structure that is formed de novo in the course of sporulation. The outer layers of the spore wall provide spores with resistance to a wide variety of environmental stresses. The major components of the outer spore wall are the polysaccharide chitosan and a polymer formed from the di-amino acid dityrosine. Though the synthesis and export pathways for dityrosine have been described, genes directly involved in dityrosine polymerization and incorporation into the spore wall have not been identified. A synthetic gene array approach to identify new genes involved in outer spore wall synthesis revealed an interconnected network influencing dityrosine assembly. This network is highly redundant both for genes of different activities that compensate for the loss of each other and for related genes of overlapping activity. Several of the genes in this network have paralogs in the yeast genome and deletion of entire paralog sets is sufficient to severely reduce dityrosine fluorescence. Solid-state NMR analysis of partially purified outer spore walls identifies a novel component in spore walls from wild type that is absent in some of the paralog set mutants. Localization of gene products identified in the screen reveals an unexpected role for lipid droplets in outer spore wall formation.

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

A visual screen of protein localization during sporulation identifies new components of prospore membrane-associated complexes in budding yeast.

During ascospore formation in Saccharomyces cerevisiae, the secretory pathway is reorganized to create new intracellular compartments, termed prospore membranes. Prospore membranes engulf the nuclei produced by the meiotic divisions, giving rise to individual spores. The shape and growth of prospore membranes are constrained by cytoskeletal structures, such as septin proteins, that associate with the membranes. Green fluorescent protein (GFP) fusions to various proteins that associate with septins at the bud neck during vegetative growth as well as to proteins encoded by genes that are transcriptionally induced during sporulation were examined for their cellular localization during prospore membrane growth. We report localizations for over 100 different GFP fusions, including over 30 proteins localized to the prospore membrane compartment. In particular, the screen identified IRC10 as a new component of the leading-edge protein complex (LEP), a ring structure localized to the lip of the prospore membrane. Localization of Irc10 to the leading edge is dependent on SSP1, but not ADY3. Loss of IRC10 caused no obvious phenotype, but an ady3 irc10 mutant was completely defective in sporulation and displayed prospore membrane morphologies similar to those of an ssp1 strain. These results reveal the architecture of the LEP and provide insight into the evolution of this membrane-organizing complex.

VPS13 regulates membrane morphogenesis during sporulation in Saccharomyces cerevisiae.

The hereditary disorders chorea acanthocytosis and Cohen syndrome are caused by mutations in different members of a family of genes that are orthologs of yeast VPS13. In vegetatively growing yeast, VPS13 is involved in the delivery of proteins to the vacuole. During sporulation, VPS13 is important for formation of the prospore membrane that encapsulates the daughter nuclei to give rise to spores. We report that VPS13 is required for multiple aspects of prospore membrane morphogenesis. VPS13 (1) promotes expansion of the prospore membrane through regulation of phosphatidylinositol phosphates, which in turn activate the phospholipase D, Spo14; (2) is required for a late step in cytokinesis that gives rise to spores; and (3) regulates a membrane-bending activity that generates intralumenal vesicles. These results demonstrate that Vps13 plays a broader role in membrane biology than previously known, which could have important implications for the functions of VPS13 orthologs in humans.

SPO71 encodes a developmental stage-specific partner for Vps13 in Saccharomyces cerevisiae.

The creation of haploid gametes in yeast, termed spores, requires the de novo formation of membranes within the cytoplasm. These membranes, called prospore membranes, enclose the daughter nuclei generated by meiosis. Proper growth and closure of prospore membranes require the highly conserved Vps13 protein. Mutation of SPO71, a meiosis-specific gene first identified as defective in spore formation, was found to display defects in membrane morphogenesis very similar to those seen in vps13Δ cells. Specifically, prospore membranes are smaller than in the wild type, they fail to close, and membrane vesicles are present within the prospore membrane lumen. As in vps13Δ cells, the levels of phophatidylinositol-4-phosphate are reduced in the prospore membranes of spo71Δ cells. SPO71 is required for the translocation of Vps13 from the endosome to the prospore membrane, and ectopic expression of SPO71 in vegetative cells results in mislocalization of Vps13. Finally, the two proteins can be coprecipitated from sporulating cells. We propose that Spo71 is a sporulation-specific partner for Vps13 and that they act in concert to regulate prospore membrane morphogenesis.

Membrane and organelle rearrangement during ascospore formation in budding yeast.

SUMMARYIn ascomycete fungi, sexual spores, termed ascospores, are formed after meiosis. Ascospore formation is an unusual cell division in which daughter cells are created within the cytoplasm of the mother cell by de novo generation of membranes that encapsulate each of the haploid chromosome sets created by meiosis. This review describes the molecular events underlying the creation, expansion, and closure of these membranes in the budding yeast, Saccharomyces cerevisiae. Recent advances in our understanding of the regulation of gene expression and the dynamic behavior of different membrane-bound organelles during this process are detailed. While less is known about ascospore formation in other systems, comparison to the distantly related fission yeast suggests that the molecular events will be broadly similar throughout the ascomycetes.

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.

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 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.

High-resolution x-ray diffraction microscopy of specifically labeled yeast cells.

X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11-13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the alpha-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane and freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy.

Pharmacological interventions for lipid transport disorders.

The recent discovery that defects in inter-organelle lipid transport are at the heart of several neurological and neurodegenerative disorders raises the challenge of identifying therapeutic strategies to correct lipid transport defects. This perspective highlights two potential strategies suggested by the study of lipid transport in budding yeast. In the first approach, small molecules are proposed that enhance the lipid transfer activity of VPS13 proteins and thereby compensate for reduced transport. In the second approach, molecules that act as inter-organelle tethers could be used to create artificial contact sites and bypass the loss of endogenous contacts.

Recruitment of the lipid kinase Mss4 to the meiotic spindle pole promotes prospore membrane formation in Saccharomyces cerevisiae.

Spore formation in the budding yeast, Saccharomyces cerevisiae, involves de novo creation of four prospore membranes, each of which surrounds a haploid nucleus resulting from meiosis. The meiotic outer plaque (MOP) is a meiosis-specific protein complex associated with each meiosis II spindle pole body (SPB). Vesicle fusion on the MOP surface creates an initial prospore membrane anchored to the SPB. Ady4 is a meiosis-specific MOP component that stabilizes the MOP-prospore membrane interaction. We show that Ady4 recruits the lipid kinase, Mss4, to the MOP. MSS4 overexpression suppresses the ady4∆ spore formation defect, suggesting that a specific lipid environment provided by Mss4 promotes maintenance of prospore membrane attachment to MOPs. The meiosis-specific Spo21 protein is an essential structural MOP component. We show that the Spo21 N terminus contains an amphipathic helix that binds to prospore membranes. A mutant in SPO21 that removes positive charges from this helix shares phenotypic similarities to ady4∆. We propose that Mss4 generates negatively charged lipids in prospore membranes that enhance binding by the positively charged N terminus of Spo21, thereby providing a mechanism by which the MOP-prospore membrane interaction is stabilized.

Membrane assembly modulates the stability of the meiotic spindle-pole body.

Spore formation in Saccharomyces cerevisiae is driven by de novo assembly of new membranes termed prospore membranes. A vesicle-docking complex called the meiosis II outer plaque (MOP) forms on the cytoplasmic faces of the spindle-pole bodies at the onset of meiosis II and serves as the initiation site for membrane formation. In this study, a fluorescence-recovery assay was used to demonstrate that the dynamics of the MOP proteins change coincident with the coalescence of precursor vesicles into a membrane. Proteins within the MOP exchange freely with a soluble pool prior to membrane assembly, but after membranes are formed they remain stably within the MOP. By contrast, constitutive spindle-pole-body proteins display low exchange in both conditions. The MOP component Ady4p plays a role in maintaining the integrity of the MOP complex, but this role differs depending on whether the MOP is associated with docked vesicles or a fully formed membrane. These results suggest an architectural rearrangement of the MOP coincident with vesicle fusion.