Biology subjects

Schuldiner, M.

Publications and source records attributed to Schuldiner, M..

48 records · Page 3Linked to original sources

bioRxiv2020

Pex14p phosphorylation modulates import of citrate synthase 2 into peroxisomes in Saccharomyces cerevisiae

The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and nonphosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes on oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Our data point to the control of the peroxisomal import of Cit2p via the state of Pex14p phosphorylation at S266, which may help S. cerevisiae cells to rapidly adjust their carbohydrate metabolism according to the nutritional conditions.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsPTSperoxisomal targeting signal;LC-MSliquid chromatography-mass spectrometry;TEVtobacco etch virus;TPATEV protease cleavage site-Protein AView Full Text

cell biology↗

bioRxiv2019

Translational regulation of Pmt1 and Pmt2 by Bfr1 affects unfolded protein O-mannosylation

O-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding GFP as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance, but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.

molecular biology↗

bioRxiv2019

AMPK regulates ESCRT-dependent microautophagy of proteasomes concomitant with proteasome storage granule assembly during glucose starvation

The ubiquitin-proteasome system regulates numerous cellular processes and is central to protein homeostasis. In proliferating yeast and many mammalian cells, proteasomes are highly enriched in the nucleus. In carbon-starved yeast, proteasomes migrate to the cytoplasm and collect in phase-separated proteasome storage granules (PSGs). PSGs dissolve and proteasomes return to the nucleus within minutes of glucose refeeding. The mechanisms by which cells regulate proteasome homeostasis under these conditions remain largely unknown. Here we show that AMP-activated protein kinase (AMPK) together with endosomal sorting complexes required for transport (ESCRTs) drive a glucose starvation-dependent microautophagy pathway that preferentially sorts aberrant proteasomes into the vacuole, thereby biasing accumulation of functional proteasomes in PSGs. The proteasome core particle (CP) and regulatory particle (RP) are regulated differently. Without AMPK, the insoluble protein deposit (IPOD) serves as an alternative site that specifically sequesters CP aggregates. Our findings reveal a novel AMPK-controlled ESCRT-mediated microautophagy mechanism in the regulation of proteasome trafficking and homeostasis under carbon starvation.

cell biology↗

bioRxiv2019

A molecular switch for Cdc48 activity and localization during oxidative stress and aging

Control over a healthy proteome begins with the birth of the polypeptide chain and ends with coordinated protein degradation. One of the major players in eukaryotic protein degradation is the essential and highly conserved ATPase, Cdc48 (p97/VCP in mammals). Cdc48 mediates clearance of misfolded proteins from the nucleus, cytosol, ER, mitochondria, and more. Here we dissect the crosstalk between cellular oxidation and Cdc48 activity by identification of a redox-sensitive site, Cys115. By integrating proteomics, biochemistry, microscopy, and bioinformatics, we show that removal of Cys115s redox-sensitive thiol group leads to accumulation of Cdc48 in the nucleus and consequently, results in severe defects in the oxidative stress response, mitochondrial fragmentation, and a decrease in ERAD and sterol biogenesis. We have thus identified a unique redox switch in Cdc48, which may provide a clearer picture of the importance of Cdc48s localization in maintaining a \"healthy\" proteome during oxidative stress and chronological aging in yeast.

molecular biology↗

bioRxiv2019

Transfer of Septin Rings to Cytokinetic Remnants Directs Age-Sensitive ER stress Surveillance Cell Cycle Re-entry

During cell division, cells must actively pass on organelles. Previously, we discovered the endoplasmic reticulum (ER) stress surveillance (ERSU) pathway that ensures the inheritance of functional ER. Activation of the ERSU causes the septin ring to mislocalize, which blocks ER inheritance and cytokinesis. Here, we found that the septin ring mislocalizes to previously utilized cell division sites called cytokinetic remnants (CRMs). The transfer of the septin ring to CRMs requires Nba1, a negative polarity component that normally prevents septin ring formation at CRMs. Furthermore, septin ring movement to CRMs relies on the ERSU component Slt2, which is recruited by binding Bem1. During ER stress, Bem1 also binds the GTP exchange factor Cdc24, without activating Cdc42, a GTPase that normally establishes polarized growth. Failure to translocate septin rings to CRMs delays the cells ability to re-enter cell division when ER homeostasis is re-established. Thus, ER stress considers the history of previous cell cycle for future cell cycle re-entry upon ER stress recovery.

cell biology↗

bioRxiv2019

Non-Canonical Regulation of Phosphatidylserine Metabolism by a Phosphatidylinositol Transfer Protein and a Phosphatidylinositol 4-OH Kinase

The phosphatidylserine (PtdSer) decarboxylase Psd2 is proposed to engage in an endoplasmic reticulum (ER)-Golgi/endosome membrane contact site (MCS) that facilitates phosphatidylserine decarboxylation to phosphatidylethanomaine (PtdEtn) in Saccharomyces cerevisiae. While this MCS is envisioned to consist of Psd2, the Sec14-like phosphatidylinositol transfer protein (PITP) Sfh4, the Stt4 phosphatidylinositol (PtdIns) 4-OH kinase, the Scs2 tether, and at least one other uncharacterized protein, functional data that address key foundations of this model are sparse. We now report that Psd2, Sfh4 and Stt4 are the only components individually required for biologically sufficient Psd2-dependent PtdEtn production. Surprisingly, neither the PtdIns-transfer activity of Sfh4 nor its capacity to activate Stt4 is required to stimulate the Psd2 pathway. Instead, Sfh4 activates the Psd2 pathway via a specific Sfh4-Psd2 physical interaction. Whereas the data indicate an Sfh4-independent association of Stt4 with Psd2 as well, we find Stt4 also regulates Psd2 activity indirectly by influencing the PtdSer pool accessible to Psd2 for decarboxylation. These collective results demonstrate that the proposed ER-Golgi/endosomal MCS model fails to provide an accurate description of the Psd2 system in yeast, and provide an example where the biological function of a Sec14-like PITP is uncoupled from its canonical activity as a PtdIns transfer protein.

cell biology↗

bioRxiv2019

Cytotoxicity of 1-deoxysphingolipid Unraveled by Genome-wide Genetic Screens and Lipidomics

Hereditary Sensory and Autonomic Neuropathy (HSAN) type IA and IC are caused by elevated levels of an atypical class of lipid named 1-deoxysphingolipid (DoxSL). How elevated levels of DoxSL perturb the physiology of the cell and how the perturbations lead to HSAN IA/C are largely unknown. In this study, we show that C26-1-deoxydihydroceramide (C26-DoxDHCer) is highly toxic to the cell, while C16- and C18-DoxDHCer are less toxic. Genome-wide genetic screens and lipidomics revealed the dynamics of DoxSL accumulation and DoxSL species responsible for the toxicity over the course of DoxSL accumulation. Furthermore, we show that disruption of F-actin organization, alteration of mitochondrial shape, and accumulation of hydrophobic bodies by DoxSL are not sufficient to cause complete cellular failure. However, combined with collapsed ER membrane, these perturbations cause cell death. Thus, we have unraveled key principles of DoxSL cytotoxicity that help to explain the clinical features of HSAN IA/C.

cell biology↗

bioRxiv2019

Disease-causing mutations in subunits of OXPHOS complex I affect their physical interactions

Mitochondrial complex I (C1) is the largest multi-subunit oxidative phosphorylation (OXPHOS) protein complex. Recent availability of a high-resolution human C1 structure, and from two non-human mammals, enabled predicting the impact of mutations on interactions involving each of the 44 C1 subunits. However, experimentally assessing the impact of the predicted interactions requires an easy and high-throughput method. Here, we created such a platform by cloning all 37 nuclear DNA (nDNA) and 7 mitochondrial DNA (mtDNA)-encoded human C1 subunits into yeast expression vectors to serve as both 'prey' and 'bait' in the split murine dihydrofolate reductase (mDHFR) protein complementation assay (PCA). We first demonstrated the capacity of this approach and then used it to examine reported pathological OXPHOS C1 mutations that occur at subunit interaction interfaces. Our results indicate that a pathological frame-shift mutation in the MT-ND2 gene, causing the replacement of 126 C-terminal residues by a stretch of only 30 amino acids, resulted in loss of specificity in ND2-based interactions involving these residues. Hence, the split mDHFR PCA is a powerful assay for assessing the impact of disease-causing mutations on pairwise protein-protein interactions in the context of a large protein complex, thus revealing the mechanism underlying any associated pathogenicity.

genomics↗

bioRxiv2019

Multiplexed electron microscopy by fluorescent barcoding allows screening for ultrastructural phenotype

Genetic screens performed using high-throughput fluorescent microscopes have generated large datasets that have contributed many insights into cell biology. However, such approaches typically cannot tackle questions requiring knowledge of ultrastructure below the resolution limit of fluorescent microscopy. Electron microscopy (EM) is not subject to this resolution limit, generating detailed images of cellular ultrastructure, but requires time consuming preparation of individual samples, limiting its throughput. Here we overcome this obstacle and describe a robust method for screening by high-throughput electron microscopy. Our approach uses combinations of fluorophores as barcodes to mark the genotype of each cell in mixed populations, and correlative light and electron microscopy to read the fluorescent barcode of each cell before it is imaged by electron microscopy. Coupled with an easy-to-use software workflow for correlation, segmentation and computer image analysis, our method allows to extract and analyze multiple cell populations from each EM sample preparation. We demonstrate the method on several organelles with samples that each contain up to 15 different yeast variants. The methodology is not restricted to yeast, can be scaled to higher-throughput, and can be utilized in multiple ways to enable electron microscopy to become a powerful screening methodology.

cell biology↗

bioRxiv2018

Systematic assessment of GFP tag position on protein localization and growth fitness in yeast

While protein tags are ubiquitously utilized in molecular biology, they harbor the potential to interfere with functional traits of their fusion counterparts. Systematic evaluation of the effect of protein tags on localization and function would promote accurate use of tags in experimental setups. Here we examine the effect of Green Fluorescent Protein (GFP) tagging at either the N or C terminus of budding yeast proteins on localization and functionality. We use a competition-based approach to decipher the relative fitness of two strains tagged on the same protein but on opposite termini and from that infer the correct, physiological localization for each protein and the optimal position for tagging. Our study provides a first of a kind systematic assessment of the effect of tags on the functionality of proteins and provides step towards broad investigation of protein fusion libraries.\n\nHighlightsO_LIProtein tags are widely used in molecular biology although they may interfere with protein function.\nC_LIO_LIThe subcellular localization of hundreds of proteins in yeast is different when tagged at the N or the C terminus.\nC_LIO_LIA competition based assay enables systematic deciphering of correct tagging terminus for essential proteins.\nC_LIO_LIThe presented approach can be used to derive physiologically relevant tagged libraries.\nC_LI

cell biology↗

bioRxiv2017

Temporal profiling of redox-dependent heterogeneity in single cells

Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe roGFP. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach we defined redox-dependent heterogeneity of yeast cells, and characterized growth, as well as proteomic and transcriptomic profiles of subpopulations of cells that differ in their redox status, but are similar in age. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

biochemistry↗

bioRxiv2017

Large-scale rewiring in a yeast hybrid

The merging of genomes in inter-specific hybrids can result in novel phenotypes, including increased growth rate and biomass yield, a phenomenon known as heterosis. We describe a budding yeast hybrid that grows faster than its parents under different environments. Phenotypically, the hybrid progresses more rapidly through cell cycle checkpoints, relieves the repression of respiration in fast growing conditions, does not slow down its growth when presented with ethanol stress, and shows increasing signs of DNA damage. A systematic genetic screen identified hundreds of alleles affecting hybrid growth whose identity vastly differed between the hybrid and its parent and between growth conditions. This large-scale rewiring of allele effects suggests that despite showing clear heterosis, the hybrid is perturbed in multiple regulatory processes. We discuss the possibility that incompatibilities contribute to hybrid vigor by perturbing safeguard mechanisms that limit growth in the parental background.

systems biology↗