Yeast Homolog Research Articles

Mitochondrial Outer Membrane Translocase MoTom20 Modulates Mitochondrial Morphology and Is Important for Infectious Growth of the Rice Blast Fungus Magnaporthe oryzae.

Mitochondria are highly dynamic organelles that constantly change their morphology to adapt to the cellular environment through fission and fusion, which is critical for a cell to maintain normal cellular functions. Despite the significance of this process in the development and pathogenicity of the rice blast fungus Magnaporthe oryzae, the underlying mechanism remains largely elusive. Here, we identified and characterized a mitochondrial outer membrane translocase, MoTom20, in M. oryzae. Targeted gene deletion revealed that MoTom20 plays an important role in vegetative growth, conidiogenesis, penetration, and infectious growth of M. oryzae. The growth rate, conidial production, appressorium turgor, and pathogenicity are decreased in the ΔMotom20 mutant compared with the wild-type and complemented strains. Further analysis revealed that MoTom20 localizes in mitochondrion and plays a key role in regulating mitochondrial fission and fusion balance, which is critical for infectious growth. Finally, we found that MoTom20 is involved in fatty-acid utilization, and its yeast homolog ScTom20 is able to rescue the defects of ΔMotom20 in mitochondrial morphology and pathogenicity. Overall, our data demonstrate that MoTom20 is a key regulator for mitochondrial morphology maintenance, which is important for infectious growth of the rice blast fungus M. oryzae. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

2-Hydroxyglutarate modulates histone methylation at specific loci and alters gene expression via Rph1 inhibition.

2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target.

Phosphorylated HP1α-Nucleosome Interactions in Phase Separated Environments.

In the nucleus, transcriptionally silent genes are sequestered into heterochromatin compartments comprising nucleosomes decorated with histone H3 Lys9 trimethylation and a protein called HP1α. This protein can form liquid-liquid droplets in vitro and potentially organize heterochromatin through a phase separation mechanism that is promoted by phosphorylation. Elucidating the molecular interactions that drive HP1α phase separation and its consequences on nucleosome structure and dynamics has been challenging due to the viscous and heterogeneous nature of such assemblies. Here, we tackle this problem by a combination of solution and solid-state NMR spectroscopy, which allows us to dissect the interactions of phosphorylated HP1α with nucleosomes in the context of phase separation. Our experiments indicate that phosphorylated human HP1α does not cause any major rearrangements to the nucleosome core, in contrast to the yeast homologue Swi6. Instead, HP1α interacts specifically with the methylated H3 tails and slows the dynamics of the H4 tails. Our results shed light on how phosphorylated HP1α proteins may regulate the heterochromatin landscape, while our approach provides an atomic resolution view of a heterogeneous and dynamic biological system regulated by a complex network of interactions and post-translational modifications.

FDX1 Is Required for the Biogenesis of Mitochondrial Cytochrome c Oxidase in Mammalian Cells

Ferredoxins (FDXs) are evolutionarily conserved iron-sulfur (Fe-S) proteins that function as electron transfer proteins in diverse metabolic pathways. Mammalian mitochondria contain two ferredoxins, FDX1 and FDX2, which share a high degree of structural similarity but exhibit different functionalities. Previous studies have established the unique role of FDX2 in the biogenesis of Fe-S clusters; however, FDX1 seems to have multiple targets in vivo, some of which are only recently emerging. Using CRISPR-Cas9-based loss-of-function studies in rat cardiomyocyte cell line, we demonstrate an essential requirement of FDX1 in mitochondrial respiration and energy production. We attribute reduced mitochondrial respiration to a specific decrease in the abundance and assembly of cytochrome c oxidase (CcO), a mitochondrial heme-copper oxidase and the terminal enzyme of the mitochondrial respiratory chain. FDX1 knockout cells have reduced levels of copper and heme a/a3, factors that are essential for the maturation of the CcO enzyme complex. Copper supplementation failed to rescue CcO biogenesis, but overexpression of heme a synthase, COX15, partially rescued COX1 abundance in FDX1 knockout cells. This finding links FDX1 function to heme a biosynthesis, and places it upstream of COX15 in CcO biogenesis like its ancestral yeast homolog. Taken together, our work has identified FDX1 as a critical CcO biogenesis factor in mammalian cells.

Maternal Embryo Effect Arrest 31 (MEE31) is a moonlighting protein involved in GDP-D-mannose biosynthesis and KAT1 potassium channel regulation

Due to anthropogenic global warming, droughts are expected to increase and water availability to decrease in the coming decades. For this reason, research is increasingly focused on developing plant varieties and crop cultivars with reduced water consumption. Transpiration occurs through stomatal pores, resulting in water loss. Potassium plays a significant role in stomatal regulation. KAT1 is an inward-rectifying potassium channel that contributes to stomatal opening. Using a yeast high-throughput screening of an Arabidopsis cDNA library, MEE31 was found to physically interact with KAT1. MEE31 was initially identified in a screen for mutants with delayed embryonic development. The gene encodes a conserved phosphomannose isomerase (PMI). We report here that MEE31 interacts with and increases KAT1 activity in yeast and this interaction was also confirmed in plants. In addition, MEE31 complements the function of the yeast homologue, whereas the truncated version recovered in the screening does not, thus uncoupling the enzymatic activity from KAT1 regulation. We show that MEE31 overexpression leads to increased stomatal opening in Arabidopsis transgenic lines. Our data suggest that MEE31 is a moonlighting protein involved in both GDP-D-mannose biosynthesis and KAT1 regulation.

Human SHQ1 variants R335C and A426V lead to severe ribosome biogenesis defects when expressed in yeast.

SHQ1 is an essential chaperone that binds the pseudouridine synthase dyskerin in the cytoplasm and escorts the enzyme to the nucleus, where dyskerin is assembled into small nucleolar RNPs (snoRNPs) of the H/ACA class. These particles carry out pseudouridine formation in ribosomal RNAs (rRNAs) and participate in maturation of rRNA precursors (pre-rRNAs). Variants of human SHQ1 have been linked to neurodevelopmental deficiencies; here we focused on two compound heterozygous mutations identified in a child showing a severe neurological disorder comprising cerebellar degeneration. To investigate the molecular defects caused by mutations R335C and A426V we used a conditional yeast strain that can be depleted of the endogenous Shq1 protein while constitutively expressing human SHQ1 (wild-type or variants). Although wild-type SHQ1 complemented the Shq1-depleted strain, cells expressing variant R335C could not support growth, and cells expressing variant A426V were temperature-sensitive. When shifted to restrictive conditions, yeast cells progressively lost H/ACA snoRNAs and accumulated unprocessed pre-rRNAs, which led to reduced production of ribosomes. Levels of Cbf5 (yeast homologue of dyskerin) were decreased in yeast cells expressing SHQ1 variants under restrictive conditions. Immunoprecipitation experiments revealed that interaction of Cbf5 with SHQ1 variants was weakened but not abolished, and yeast two-hybrid assays showed that mutation R335C is more deleterious than mutation A426V. Our data provide additional evidence for the critical role of SHQ1 in chaperoning the pseudouridine synthase dyskerin, and how its inadequate function has detrimental consequences on the production of H/ACA snoRNPs and ribosomes.

LAMMER Kinase Modulates Cell Cycle by Phosphorylating the MBF Repressor, Yox1, in Schizosaccharomyces pombe

Lkh1, a LAMMER kinase homolog in the fission yeast Schizosaccharomyces pombe, acts as a negative regulator of filamentous growth and flocculation. It is also involved in the response to oxidative stress. The lkh1-deletion mutant displays slower cell growth, shorter cell size, and abnormal DNA content compared to the wild type. These phenotypes suggest that Lkh1 controls cell size and cell cycle progression. When we performed microarray analysis using the lkh1-deletion mutant, we found that only four of the up-regulated genes in the lkh1-deletion were associated with the cell cycle. Interestingly, all of these genes are regulated by the Mlu1 cell cycle box binding factor (MBF), which is a transcription complex responsible for regulating the expression of cell cycle genes during the G1/S phase. Transcription analyses of the MBF-dependent cell-cycle genes, including negative feedback regulators, confirmed the up-regulation of these genes by the deletion of lkh1. Pull-down assay confirmed the interaction between Lkh1 and Yox1, which is a negative feedback regulator of MBF. This result supports the involvement of LAMMER kinase in cell cycle regulation by modulating MBF activity. In vitro kinase assay and NetPhosK 2.0 analysis with the Yox1T40,41A mutant allele revealed that T40 and T41 residues are the phosphorylation sites mediated by Lkh1. These sites affect the G1/S cell cycle progression of fission yeast by modulating the activity of the MBF complex.

Divergent roles of Rad4 and Rad23 homologs in Metarhizium robertsii's resistance to solar ultraviolet damage.

The anti-ultraviolet (UV) role of a Rad4-Rad23-Rad33 complex in budding yeast relies on nucleotide excision repair (NER), which is mechanistically distinct from photorepair of DNA lesions generated under solar UV irradiation but remains poorly known in filamentous fungi. Here, two nucleus-specific Rad4 paralogs (Rad4A and Rad4B) and nucleocytoplasmic shuttling Rad23 ortholog are functionally characterized by multiple analyses of their null mutants in Metarhizium robertsii, an entomopathogenic fungus lacking Rad33. Rad4A was proven to interact with Rad23 and contribute significantly more to conidial UVB resistance (90%) than Rad23 (65%). Despite no other biological function, Rad4A exhibited a very high activity in photoreactivation of UVB-impaired/inactivated conidia by 5-h light exposure due to its interaction with Rad10, an anti-UV protein clarified previously to have acquired a similar photoreactivation activity through its interaction with a photolyase in M. robertsii. The NER activity of Rad4A or Rad23 was revealed by lower reactivation rates of moderately impaired conidia after 24-h dark incubation but hardly observable at the end of 12-h dark incubation, suggesting an infeasibility of its NER activity in the field where nighttime is too short. Aside from a remarkable contribution to conidial UVB resistance, Rad23 had pleiotropic effect in radial growth, aerial conidiation, antioxidant response, and cell wall integrity but no photoreactivation activity. However, Rad4B proved redundant in function. The high photoreactivation activity of Rad4A unveils its essentiality for M. robertsii's fitness to solar UV irradiation and is distinct from the yeast homolog's anti-UV role depending on NER. IMPORTANCE Resilience of solar ultraviolet (UV)-impaired cells is crucial for the application of fungal insecticides based on formulated conidia. Anti-UV roles of Rad4, Rad23, and Rad33 rely upon nucleotide excision repair (NER) of DNA lesions in budding yeast. Among two Rad4 paralogs and Rad23 ortholog characterized in Metarhizium robertsii lacking Rad33, Rad4A contributes to conidial UVB resistance more than Rad23, which interacts with Rad4A rather than functionally redundant Rad4B. Rad4A acquires a high activity in photoreactivation of conidia severely impaired or inactivated by UVB irradiation through its interaction with Rad10, another anti-UV protein previously proven to interact with a photorepair-required photolyase. The NER activity of either Rad4A or Rad23 is seemingly extant but unfeasible under field conditions. Rad23 has pleiotropic effect in the asexual cycle in vitro but no photoreactivation activity. Therefore, the strong anti-UV role of Rad4A depends on photoreactivation, unveiling a scenario distinct from the yeast homolog's NER-reliant anti-UV role.

Kar4 is required for the normal pattern of meiotic gene expression.

Kar4p, the yeast homolog of the mammalian methyltransferase subunit METTL14, is required for efficient mRNA m6A methylation, which regulates meiotic entry. Kar4p is also required for a second seemingly non-catalytic function during meiosis. Overexpression of the early meiotic transcription factor, IME1, can bypass the requirement for Kar4p in meiotic entry but the additional overexpression of the translational regulator, RIM4, is required to permit sporulation in kar4Δ/Δ. Using microarray analysis and RNA sequencing, we sought to determine the impact of removing Kar4p and consequently mRNA methylation on the early meiotic transcriptome in a strain background (S288c) that is sensitive to the loss of early meiotic regulators. We found that kar4Δ/Δ mutants have a largely wild type transcriptional profile with the exception of two groups of genes that show delayed and reduced expression: (1) a set of Ime1p-dependent early genes as well as IME1, and (2) a set of late genes dependent on the mid-meiotic transcription factor, Ndt80p. The early gene expression defect is likely the result of the loss of mRNA methylation and is rescued by overexpressing IME1, but the late defect is only suppressed by overexpression of both IME1 and RIM4. The requirement for RIM4 led us to predict that the non-catalytic function of Kar4p, like methyltransferase complex orthologs in other systems, may function at the level of translation. Mass spectrometry analysis identified several genes involved in meiotic recombination with strongly reduced protein levels, but with little to no reduction in transcript levels in kar4Δ/Δ after IME1 overexpression. The low levels of these proteins were rescued by overexpression of RIM4 and IME1, but not by the overexpression of IME1 alone. These data expand our understanding of the role of Kar4p in regulating meiosis and provide key insights into a potential mechanism of Kar4p's later meiotic function that is independent of mRNA methylation.

Kar4, the yeast homolog of METTL14, is required for mRNA m6A methylation and meiosis.

KAR4, the yeast homolog of the mammalian mRNA N6A-methyltransferase complex component METTL14, is required for two disparate developmental programs in Saccharomyces cerevisiae: mating and meiosis. To understand KAR4's role in yeast mating and meiosis, we used a genetic screen to isolate 25 function-specific mutant alleles, which map to non-overlapping surfaces on a predicted structure of the Kar4 protein (Kar4p). Most of the mating-specific alleles (Mat-) abolish Kar4p's interaction with the transcription factor Ste12p, indicating that Kar4p's mating function is through Ste12p. In yeast, the mRNA methyltransferase complex was previously defined as comprising Ime4p (Kar4p's paralog and the homolog of mammalian METTL3), Mum2p (homolog of mammalian WTAP), and Slz1p (MIS), but not Kar4p. During meiosis, Kar4p interacts with Ime4p, Mum2p, and Slz1p. Moreover, cells lacking Kar4p have highly reduced levels of mRNA methylation during meiosis indicating that Kar4p is a key member of the methyltransferase complex, as it is in humans. Analysis of kar4Δ/Δ and 7 meiosis-specific alleles (Mei-) revealed that Kar4p is required early in meiosis, before initiation of S-phase and meiotic recombination. High copy expression of the meiotic transcriptional activator IME1 rescued the defect of these Mei- alleles. Surprisingly, Kar4p was also found to be required at a second step for the completion of meiosis and sporulation. Over-expression of IME1 in kar4Δ/Δ permits pre-meiotic S-phase, but most cells remained arrested with a monopolar spindle. Analysis of the function-specific mutants revealed that roughly half became blocked after premeiotic DNA synthesis and did not sporulate (Spo-). Loss of Kar4p's Spo function was suppressed by overexpression of RIM4, a meiotic translational regulator. Overexpression of IME1 and RIM4 together allowed sporulation of kar4Δ/Δ cells. Taken together, these data suggest that Kar4p regulates meiosis at multiple steps, presumably reflecting requirements for methylation in different stages of meiotic gene expression.

Electroclinical Features in Two Novel STRADA Patients and a Functional Yeast Assay for the Validation of Missense STRADA Mutations

Loss of function of the STRADA gene, an upstream mTOR inhibitor, causes a rare neurodevelopmental disorder characterized by polyhydramnios, megalencephaly, and symptomatic epilepsy (PMSE syndrome). Patients display a homogeneous phenotype including early-onset drug-resistant epilepsy, severe psychomotor delay, multisystemic comorbidities, and increased risk of premature death. The administration of sirolimus, an mTOR inhibitor, is helpful in controlling seizures in this syndrome. We report the electroclinical phenotype of two novel patients and the development of a yeast model to validate the pathogenicity of missense variants. Patient 1 harbored a missense STRADA variant and had a peculiar electroclinical phenotype with a relatively mild epilepsy course. Patient 2 harbored a truncating STRADA variant and showed a typical PMSE phenotype and a favorable response to early treatment with sirolimus.When we modeled the p.(Ser264Arg) STRADA change in its yeast homolog SPS1, it impaired SPS1 function.The results underlie the importance of a timely molecular diagnosis in these patients and show that yeast is a simple yet effective model to validate the pathogenicity of missense variants.

Circadian clock control of tRNA synthetases in Neurospora crassa.

Background: In Neurospora crassa, the circadian clock controls rhythmic mRNA translation initiation through regulation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2). Active CPC-3 phosphorylates and inactivates eIF2α, leading to higher phosphorylated eIF2α (P-eIF2α) levels and reduced translation initiation during the subjective day. This daytime activation of CPC-3 is driven by its binding to uncharged tRNA, and uncharged tRNA levels peak during the day under control of the circadian clock. The daily rhythm in uncharged tRNA levels could arise from rhythmic amino acid levels or aminoacyl-tRNA synthetase (aaRSs) levels. Methods: To determine if and how the clock potentially controls rhythms in aspartyl-tRNA synthetase (AspRS) and glutaminyl-tRNA synthetase (GlnRS), both observed to be rhythmic in circadian genomic datasets, transcriptional and translational fusions to luciferase were generated. These luciferase reporter fusions were examined in wild type (WT), clock mutant Δ frq, and clock-controlled transcription factor deletion strains. Results: Translational and transcriptional fusions of AspRS and GlnRS to luciferase confirmed that their protein levels are clock-controlled with peak levels at night. Moreover, clock-controlled transcription factors NCU00275 and ADV-1 drive robust rhythmic protein expression of AspRS and GlnRS, respectively. Conclusions: These data support a model whereby coordinate clock control of select aaRSs drives rhythms in uncharged tRNAs, leading to rhythmic CPC-3 activation, and rhythms in translation of specific mRNAs.

The Histone Deacetylase HstD Regulates Fungal Growth, Development and Secondary Metabolite Biosynthesis in Aspergillus terreus.

Histone acetylation modification significantly affects secondary metabolism in filamentous fungi. However, how histone acetylation regulates secondary metabolite synthesis in the lovastatin (a lipid-lowering drug) producing Aspergillus terreus remains unknown because protein is involved and has been identified in this species. Here, the fungal-specific histone deacetylase gene, hstD, was characterized through functional genomics in two marine-derived A. terreus strains, Mj106 and RA2905. The results showed that the ablation of HstD resulted in reduced mycelium growth, less conidiation, and decreased lovastatin biosynthesis but significantly increased terrein biosynthesis. However, unlike its homologs in yeast, HstD was not required for fungal responses to DNA damage agents, indicating that HstD likely plays a novel role in the DNA damage repair process in A. terreus. Furthermore, the loss of HstD resulted in a significant upregulation of H3K56 and H3K27 acetylation when compared to the wild type, suggesting that epigenetic functions of HstD, as a deacetylase, target H3K27 and H3K56. Additionally, a set of no-histone targets with potential roles in fungal growth, conidiation, and secondary metabolism were identified for the first time using acetylated proteomic analysis. In conclusion, we provide a comprehensive analysis of HstD for its targets in histone or non-histone and its roles in fungal growth and development, DNA damage response, and secondary metabolism in A. terreus.

Adenine nucleotide carrier protein dysfunction in human disease.

Adenine nucleotide translocase (ANT) is the prototypical member of the mitochondrial carrier protein family, primarily involved in ADP/ATP exchange across the inner mitochondrial membrane. Several carrier proteins evolutionarily related to ANT, including SLC25A24 and SLC25A25, are believed to promote the exchange of cytosolic ATP-Mg2+ with phosphate in the mitochondrial matrix. They allow a net accumulation of adenine nucleotides inside mitochondria, which is essential for mitochondrial biogenesis and cell growth. In the last two decades, mutations in the heart/muscle isoform 1 of ANT (ANT1) and the ATP-Mg2+ transporters have been found to cause a wide spectrum of human diseases by a recessive or dominant mechanism. Although loss-of-function recessive mutations cause a defect in oxidative phosphorylation and an increase in oxidative stress which drives the pathology, it is unclear how the dominant missense mutations in these proteins cause human diseases. In this review, we focus on how yeast was productively used as a model system for the understanding of these dominant diseases. We also describe the relationship between the structure and function of ANT and how this may relate to various pathologies. Particularly, mutations in Aac2, the yeast homolog of ANT, were recently found to clog the mitochondrial protein import pathway. This leads to mitochondrial precursor overaccumulation stress (mPOS), characterized by the toxic accumulation of unimported mitochondrial proteins in the cytosol. We anticipate that in coming years, yeast will continue to serve as a useful model system for the mechanistic understanding of mitochondrial protein import clogging and related pathologies in humans.

Mechanism of RanGTP priming H2A-H2B release from Kap114 in an atypical RanGTP•Kap114•H2A-H2B complex

Previously, we showed that the nuclear import receptor Importin-9 wraps around the H2A-H2B core to chaperone and transport it from the cytoplasm to the nucleus. However, unlike most nuclear import systems where RanGTP dissociates cargoes from their importins, RanGTP binds stably to the Importin-9•H2A-H2B complex, and formation of the ternary RanGTP•Importin-9•H2A-H2B complex facilitates H2A-H2B release to the assembling nucleosome. It was unclear how RanGTP and the cargo H2A-H2B can bind simultaneously to an importin, and how interactions of the three components position H2A-H2B for release. Here, we show cryo-EM structures of Importin-9•RanGTP and of its yeast homolog Kap114, including Kap114•RanGTP, Kap114•H2A-H2B, and RanGTP•Kap114•H2A-H2B, to explain how the conserved Kap114 binds H2A-H2B and RanGTP simultaneously and how the GTPase primes histone transfer to the nucleosome. In the ternary complex, RanGTP binds to the N-terminal repeats of Kap114 in the same manner as in the Kap114/Importin-9•RanGTP complex, and H2A-H2B binds via its acidic patch to the Kap114 C-terminal repeats much like in the Kap114/Importin-9•H2A-H2B complex. Ran binds to a different conformation of Kap114 in the ternary RanGTP•Kap114•H2A-H2B complex. Here, Kap114 no longer contacts the H2A-H2B surface proximal to the H2A docking domain that drives nucleosome assembly, positioning it for transfer to the assembling nucleosome or to dedicated H2A-H2B chaperones in the nucleus.

Transcriptional repression by the histone tails in budding yeast is mediated by Rpd3, Tup1-Ssn6, and Bur6/NC2

Chromatin-mediated transcriptional regulation is modulated by post-translational modifications of the core histones, particularly the H3 and H4 unstructured amino termini, or “tails”. In budding yeast, the H3 and H4 tails can be deacetylated by Rpd3 to repress specific target genes, and hypoacetylated histones can facilitate recruitment of the Tup1-Ssn6 complex to effect gene repression. However, the extent to which these mechanisms are used to effect repression by the histone tails, and whether other factors similarly collaborate with the tails to facilitate gene repression, has not been determined. Here, a chromatin modifier compendium of 170 gene expression profiles from yeast strains mutated for chromatin-related genes was used to query the effect of the corresponding mutations on gene cohorts repressed by the histone H3 and H4 tails and/or by Rpd3. The resulting analysis reveals that repression of nearly all of the genes repressed by the histone tails requires Rpd3 and/or the Tup1-Ssn6 complex. Repression by Rpd3 occurs via the Rpd3-L complex, and TFIID-dominated genes are underrepresented among genes repressed by mutations or deletions of the H3 or H4 tails, in accord with previous work. In addition, Bur6, the yeast homolog of human NC2α, is required for repression at ∼50 % of genes repressed by the H3 or H4 tail. These results illuminate genome-wide repression mechanisms utilized by the histone tails in yeast and raise new questions regarding the role of Bur6 in histone tail-mediated repression and whether parallels exist in metazoan cells.

Distinct Interaction Modes for the Eukaryotic RNA Polymerase Alpha-like Subunits.

Eukaryotic DNA-dependent RNA polymerases (Pols I-III) encode two distinct alpha-like heterodimers where one is shared between Pols I and III, and the other is unique to Pol II. Human alpha-like subunit mutations are associated with several diseases including Treacher Collins Syndrome (TCS), 4H leukodystrophy, and primary ovarian sufficiency. Yeast is commonly used to model human disease mutations, yet it remains unclear whether the alpha-like subunit interactions are functionally similar between yeast and human homologs. To examine this, we mutated several regions of the yeast and human small alpha-like subunits and used biochemical and genetic assays to establish the regions and residues required for heterodimerization with their corresponding large alpha-like subunits. Here we show that different regions of the small alpha-like subunits serve differential roles in heterodimerization, in a polymerase- and species-specific manner. We found that the small human alpha-like subunits are more sensitive to mutations, including a "humanized" yeast that we used to characterize the molecular consequence of the TCS-causingPOLR1D G52E mutation. These findings help explain why some alpha subunit associated disease mutations have little to no effect when made in their yeast orthologs and offer a better yeast model to assess the molecular basis of POLR1D associated disease mutations.

Abasic site-peptide cross-links are blocking lesions repaired by AP endonucleases.

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

Ceramide synthase homolog Tlc4 maintains nuclear envelope integrity via its Golgi translocation.

Maintaining the integrity of the nuclear envelope (NE) is essential for preventing genomic DNA damage. Recent studies have shown that enzymes that catalyze lipid synthesis are involved in NE maintenance, but the underlying mechanism remains unclear. Here, we found that the ceramide synthase (CerS) homolog in the fission yeast Schizosaccharomyces pombe Tlc4 (SPAC17A2.02c) suppressed NE defects in cells lacking the NE proteins Lem2 and Bqt4. Tlc4 possesses a TRAM/LAG1/CLN8-domain conserved in CerS proteins and functions through its non-catalytic activity. Tlc4 was localized at the NE and ER, similar to CerS proteins, and also showed unique additional localization at the cis- and medial-Golgi cisternae. Growth and mutation analyses revealed that Golgi localization of Tlc4 was tightly linked to its activity of suppressing the defects in the double-deletion mutant of Lem2 and Bqt4. Our results suggest that Lem2 and Bqt4 control the translocation of Tlc4 from the NE to the Golgi, which is necessary for maintaining NE integrity.

Mitochondrial protein import clogging as a mechanism of disease.

Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in ADP/ATP translocase 1 (ANT1), and its yeast homolog ADP/ATP carrier 2 (Aac2), cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis, and cell viability independent of ANT1's nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/ANT1 causes severe clogging primarily at the translocase of the outer membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger ANT1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy ANT1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.