Biology subjects

Brouwers, N.

Publications and source records attributed to Brouwers, N..

14 recordsLinked to original sources

bioRxiv2025

p125A (Sec23ip) couples COPII coat assembly with donor-acceptor membrane organization to facilitate tunnel-based traffic.

COPII-coat proteins play a crucial role in generating small vesicles at endoplasmic reticulum (ER) exit sites (ERES). However, they also assemble at the necks of tunnels and tubules connecting the ER exit site (donor) to the ERGIC/cis-Golgi cisterna (acceptor). How can COPII support these two traffic mechanisms? Through cell-free reconstitutions, we have found that the apposition of donor and acceptor membranes is important for the assembly of the outer layer of the COPII coat (Sec13/31) but had minimal impact on Sar1-induced COPII inner layer (Sec23/24) recruitment. The expression of the adaptor protein p125A, which binds to phosphatidylinositol 4-phosphate (PI4P), Sec31 and Sec23, stabilized the contact between the donor and acceptor membranes and promoted COPII outer layer assembly. A p125A-chimera expressing a Golgi-targeted PI4P-binding domain could also support outer layer assembly. Notably, the C-terminal helical domain of Sec31A, which interacts with p125A, was essential for its assembly at ERES. In cells lacking p125A, the assembly of the COPII outer layer was selectively destabilized. Transcriptome and secretome analyses reveal selective adjustments to extracellular matrix remodeling and collagen secretion, which corresponded with selective inhibition of fibrillar collagen traffic from the ER. Thus, p125A connects the outer COPII layer at ERES with PI4P-rich ERGIC/cis-Golgi membranes, coordinating COPII assembly with tunnel-driven collagen traffic.

cell biology↗

bioRxiv2022

Defects in lipid homeostasis reflect the function of TANGO2 in Acyl-CoA metabolism

We show that TANGO2, which lacks a transmembrane domain localizes predominantly to mitochondria and transiently to endoplasmic reticulum (ER) and lipid droplets (LDs). Evaluation of lipids in HepG2 cells lacking TANGO2 revealed an increase in the size of lipid droplets and reactive oxygen species production. There is also a marked increase lysophosphatidic acid (LPA) and a concomitant decrease in its biosynthetic precursor phosphatidic acid (PA). These changes are exacerbated in nutrient starved cells. Based on our data, we suggest that the principle function of TANGO2 is in acyl-CoA metabolism, which is necessary for the acylation of LPA to generate PA. This defect subsequently affects metabolism of many other fatty acids. These data help explain the physiological consequence of TANGO2 that induce acute metabolic crisis including rhabdomyolysis, cardiomyopathy and cardiac arrhythmias often leading to fatality upon starvation and stress.

cell biology↗

bioRxiv2022

Biphasic release propensity of mucin granules is supervised by TSPAN8

Agonist-mediated stimulated pathway of mucin and insulin release is biphasic in which a rapid fusion of pre-docked granules is followed by slow docking and fusion of granules from the reserve pool. The sustained neurotransmitter release also necessitates docking of vesicles from a reserve pool. We present here a surprising finding that plasma membrane-located tetraspanin-8 (Tspan-8) sequesters syntaxin-2 (Stx2) to control external agonist-dependent mucin release. Tspan-8 specifically affects fusion of granules in reserve during the second phase of stimulated mucin release. The Tspan-8 and Stx2 complex does not contain VAMP-8 and Munc18, which are required for fusion of mucin granules. We suggest that by sequestering Stx2, Tspan-8 prevents docking granules in the reserve pool. In the absence of Tspan-8, granules in reserve pool are free to dock to Stx2 and their fusion doubles the quantities of mucins secreted. Tspan-8 thus emerges as the long-sought component that controls biphasic mucin release. We suggest a similar mechanism likely controls biphasic insulin and sustained neurotransmitter release.

cell biology↗

bioRxiv2021

The ulcerative colitis associated gene FUT8 regulates the quantity and quality of secreted mucins

Fucosylation of mucins, the main macrocomponents of the mucus layer that protects the digestive tract from pathogens, increases their viscoelasticity and shear stress resistance. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here we show that the levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients compared to normal individuals. Overexpressing FUT8, as observed in UC, in mucin-producing HT29-18N2 colonic cell line increases trafficking of MUC1 to plasma membrane and secretion of MUC2/MUC5AC. FUT8 depletion (FUT8 KD), instead, causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to shear stress compared to mucins secreted by FUT8 KD cells. These data fit well with the Fut8-/- mice phenotype, which are protected against UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins compared to Fut8+/+ mice. Together, these data reveal that FUT8 optimizes the viscoelastic properties of the extracellular mucous by controlling the quantities of mucins secreted. SIGNIFICANCE STATEMENTMucins, the major components of the mucous barrier that protects our body from pathogens, are heavily glycosylated proteins. Changes their amounts and properties will alter the viscosity of mucous. Here we show that FUT8, a glycosylation enzyme of the Golgi apparatus, can control the viscosity of secreted mucins. Mucin secreting cells of the distal colon express FUT8, but their levels are altered in Ulcerative colitis patients. As a result, mucous produced by these cells is easily washed away, which exposes them to pathogens. We suggest that a defective mucous production is the main cause of initial inflammation observed in disease. Our findings help in understanding how cells control the quality of mucins and provide a means to prevent Ulcerative colitis.

cell biology↗

bioRxiv2021

Building new compartments for unconventional protein secretion from the early and late Golgi membranes

CUPS, a compartment for unconventional secretion of signal sequence lacking proteins, is built during starvation. CUPS, lacking the Golgi specific glycosyltransferases, form by COPI independent extraction of membranes from the early Golgi cisterna, require PI4P for their biogenesis and PI3P for stability. We now show that a PI4P effector Drs2 of the trans-Golgi network, relocates to a new compartment monikered TCUPS because it touches CUPS. Although localized to TCUPS, Drs2 is required for CUPS formation specifically by interacting with Rcy1, and this process is essential for unconventional secretion. Visualizing cells by 4D SCLIM technology revealed that tubules emanating from TCUPS are often collared by CUPS and severed. Incidentally, while CUPS are stable, TCUPS are vesiculated at late stages of starvation. This mirrors the dynamics of the early and late Golgi during conventional protein secretion. TCUPS and CUPS thus emerge as the functional equivalent of early and late Golgi of the conventional secretory pathway, thus representing key compartments in unconventional secretion.

cell biology↗

bioRxiv2020

Palmitoylation of KChIP3 controls baseline mucin secretion

Baseline mucin secretion (BMS) is independent of external agonists and controlled by a small calcium binding protein named KChIP3. KChIP3-hosting mucin granules are not released until intracellular cytosolic calcium oscillations reach a threshold, KChIP3 binds calcium and detaches from granules, allowing their fusion to plasma membrane. Loss of KChIP3 or blocking its membrane attachment causes mucin hypersecretion. How is KChIP3 recruited to mucin granules? We show here that zDHHC (aspartate-histidine-histidine-cysteine motif in a cysteine-rich, zinc finger-like domain) S-acyl-transferase dependent palmitoylation modulates binding of KChIP3 to mucin granules thereby affecting mucin secretion. We have found that inhibiting zDHHC-mediated palmitoylation in differentiated HT29-18N2, which express the Golgi-localized zDHHC3 and zDHHC4, releases KChIP3 from mucin granules and increases baseline mucin secretion. Mutation of the palmitoylation sites in KChIP3 (Cysteines 122 and 123 to Alanine) quantitatively reduces its attachment to mucin granules. Expression of KChIP3-WT in HT29-18N2 cell lines stably depleted of KChIP3 inhibits mucin secretion, whereas expression of non-palmitoylated KChIP3 (KChIP3-AA) only partially rescues the effect of KChIP3 depletion and the cells maintain higher levels of baseline secretion compared to KChIP3-WT cells. Altogether, our data suggest that zDHHC3 or zDHHC4-dependent palmitoylation is involved in KChIP3 recruitment to mucin granules to control the baseline mucin secretion.

cell biology↗

bioRxiv2019

Maltotriose consumption by hybrid Saccharomyces pastorianus is heterotic and results from regulatory cross-talk between parental sub-genomes

S. pastorianus strains are hybrids of S. cerevisiae and S. eubayanus that have been domesticated for several centuries in lager-beer brewing environments. As sequences and structures of S. pastorianus genomes are being resolved, molecular mechanisms and evolutionary origin of several industrially relevant phenotypes remain unknown. This study investigates how maltotriose metabolism, a key feature in brewing, may have arisen in early S. eubayanus x S. cerevisiae hybrids. To address this question, we generated a near-complete genome assembly of Himalayan S. eubayanus strains of the Holarctic subclade. This group of strains have been proposed to be the origin of the S. eubayanus subgenome of current S. pastorianus strains. The Himalayan S. eubayanus genomes harbored several copies of a SeAGT1 -oligoglucoside transporter gene with high sequence identity to genes encountered in S. pastorianus. Although Himalayan S. eubayanus strains are unable to grown on maltose and maltotriose, their maltose-hydrolase and SeMALT1 and SeAGT1 maltose-transporter genes complemented the corresponding null mutants of S. cerevisiae. Expression, in a Himalayan S. eubayanus strain, of a functional S. cerevisiae maltose-metabolism regulator gene (MALx3) enabled growth on oligoglucosides. The hypothesis that the maltotriose-positive phenotype in S. pastorianus is a result of heterosis was experimentally tested by constructing a S. cerevisiae x S. eubayanus laboratory hybrid with a complement of maltose-metabolism genes that resembles that of current S. pastorianus strains. The ability of this hybrid to consume maltotriose in brewers wort demonstrated regulatory cross talk between sub-genomes and thereby validated this hypothesis. These results provide experimental evidence of the evolutionary origin of an essential phenotype of lager-brewing strains and valuable knowledge for industrial exploitation of laboratory-made S. pastorianus-like hybrids.\n\nImportanceS.pastorianus, a S.cerevisiae X S.eubayanus hybrid, is used for production of lager beer, the most produced alcoholic beverage worldwide It emerged by spontaneous hybridization and have colonized early lager-brewing processes. Despite accumulation and analysis of genome sequencing data of S.pastorianus parental genomes, the genetic blueprint of industrially relevant phenotypes remains unknown. Assimilation of wort abundant sugar maltotriose has been postulated to be inherited from S.cerevisiae parent. Here, we demonstrate that although Asian S.eubayanus isolates harbor a functional maltotriose transporter SeAGT1 gene, they are unable to grow on -oligoglucosides, but expression of S. cerevisae regulator ScMAL13 was sufficient to restore growth on trisaccharides. We hypothesized that S. pastorianus maltotriose phenotype results from regulatory interaction between S.cerevisae maltose transcription activator and the promoter of SeAGT1. We experimentally confirmed the heterotic nature of the phenotype and thus this results provide experimental evidence of the evolutionary origin of an essential phenotype of lager-brewing strains.

microbiology↗

bioRxiv2019

Reactive oxygen species (ROS) production triggers unconventional secretion of antioxidant enzymes

Nutrient deprivation triggers the release of signal-sequence-lacking Acb1 and the antioxidant superoxide dismutase 1 (SOD1). We now report that secreted SOD1 is functionally active and accompanied by export of other antioxidant enzymes such as thioredoxins, (Trx1 and Trx2) and peroxiredoxin Ahp1, in a Grh1 dependent manner. Our data reveal that starvation leads to production of non-toxic levels of reactive oxygen species (ROS). Treatment of cells with N-acetylcysteine (NAC), which sequesters ROS, prevents antioxidants and Acb1 secretion. Starved cells lacking Grh1 are metabolically active, but defective in their ability to regrow upon return to growth conditions. Treatment with NAC restored the Grh1 dependent effect of starvation on cell growth. In sum, starvation triggers ROS production and cells respond by secreting antioxidants and Acb1, which is an important lipogenic signaling molecule in mammalian cells. We suggest that unconventional secretion of antioxidants and Acb1 like activities maintains cells in a form necessary for growth upon their eventual return from starvation to normal conditions.

cell biology↗

bioRxiv2019

Nanopore sequencing and comparative genome analysis confirm lager-brewing yeasts originated from a single hybridization

BackgroundThe lager brewing yeast, S. pastorianus, is a hybrid between S. cerevisiae and S. eubayanus with extensive chromosome aneuploidy. S. pastorianus is subdivided into Group 1 and Group 2 strains, where Group 2 strains have higher copy number and a larger degree of heterozygosity for S. cerevisiae chromosomes. As a result, Group 2 strains were hypothesized to have emerged from a hybridization event distinct from Group 1 strains. Current genome assemblies of S. pastorianus strains are incomplete and highly fragmented, limiting our ability to investigate their evolutionary history.\n\nResultsTo fill this gap, we generated a chromosome-level genome assembly of the S. pastorianus strain CBS 1483 using MinION sequencing and analysed the newly assembled subtelomeric regions and chromosome heterozygosity. To analyse the evolutionary history of S. pastorianus strains, we developed Alpaca: a method to compute sequence similarity between genomes without assuming linear evolution. Alpaca revealed high similarities between the S. cerevisiae subgenomes of Group 1 and 2 strains, and marked differences from sequenced S. cerevisiae strains.\n\nConclusionsOur findings suggest that Group 1 and Group 2 strains originated from a single hybridization involving a heterozygous S. cerevisiae strain, followed by different evolutionary trajectories. The clear differences between both groups may originate from a severe population bottleneck caused by the isolation of the first pure cultures. Alpaca provides a computationally inexpensive method to analyse evolutionary relationships while considering non-linear evolution such as horizontal gene transfer and sexual reproduction, providing a complementary viewpoint beyond traditional phylogenetic approaches.

bioinformatics↗

bioRxiv2019

New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathways function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to the Golgi complex. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion, and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.

cell biology↗

bioRxiv2018

Laboratory evolution of a Saccharomyces cerevisiae x S. eubayanus hybrid under simulated lager-brewing conditions: genetic diversity and phenotypic convergence

Saccharomyces pastorianus lager-brewing yeasts are domesticated hybrids of S. cerevisiae x S. eubayanus that display extensive inter-strain chromosome copy number variation and chromosomal recombinations. It is unclear to what extent such genome rearrangements are intrinsic to the domestication of hybrid brewing yeasts and whether they contribute to their industrial performance. Here, an allodiploid laboratory hybrid of S. cerevisiae and S. eubayanus was evolved for up to 418 generations on wort under simulated lager-brewing conditions in six independent sequential batch bioreactors. Characterization of 55 single-cell isolates from the evolved cultures showed large phenotypic diversity and whole-genome sequencing revealed a large array of mutations. Frequent loss of heterozygosity involved diverse, strain-specific chromosomal translocations, which differed from those observed in domesticated, aneuploid S. pastorianus brewing strains. In contrast to the extensive aneuploidy of domesticated S. pastorianus strains, the evolved isolates only showed limited (segmental) aneuploidy. Specific mutations could be linked to calcium-dependent flocculation, loss of maltotriose utilisation and loss of mitochondrial activity, three industrially relevant traits that also occur in domesticated S. pastorianus strains. This study indicates that fast acquisition of extensive aneuploidy is not required for genetic adaptation of S. cerevisiae x S. eubayanus hybrids to brewing environments. In addition, this work demonstrates that, consistent with the diversity of brewing strains for maltotriose utilization, domestication under brewing conditions can result in loss of this industrially relevant trait. These observations have important implications for the design of strategies to improve industrial performance of novel laboratory-made hybrids.

evolutionary biology↗

bioRxiv2018

In vivo recombination of Saccharomyces eubayanus maltose-transporter genes yields a chimeric transporter that enables maltotriose fermentation

Saccharomyces pastorianus lager-brewing yeasts are aneuploid S. cerevisiae x S. eubayanus hybrids, whose genomes have been shaped by domestication in brewing-related contexts. In contrast to most S. cerevisiae and S. pastorianus strains, S. eubayanus cannot utilize maltotriose, a major carbohydrate in brewers wort. Accordingly, S. eubayanus CBS 12357{top} harbors four SeMALT maltose-transporter genes, but no genes resembling the S. cerevisiae maltotriose-transporter gene ScAGT1 or the S. pastorianus maltotriose-transporter gene SpMTY1. To study the evolvability of maltotriose utilization in S. eubayanus CBS 12357{top}, maltotriose-assimilating mutants obtained after UV mutagenesis were subjected to laboratory evolution in carbon-limited chemostat cultures on maltotriose-enriched wort. An evolved strain showed improved maltose and maltotriose fermentation, as well as an improved flavor profile, in 7-L fermenter experiments on industrial wort. Whole-genome sequencing revealed a novel mosaic SeMALT413 gene, resulting from repeated gene introgressions by non-reciprocal translocation of at least three SeMALT genes. The predicted tertiary structure of SeMalt413 was comparable to the original SeMalt transporters, but overexpression of SeMALT413 sufficed to enable growth on maltotriose, indicating gene neofunctionalization had occurred. The mosaic structure of SeMALT413 resembles the structure of S. pastorianus maltotriose-transporter gene SpMTY1, which has sequences with high similarity to alternatingly ScMALx1 and SeMALT3. Evolution of the maltotriose-transporter landscape in hybrid S. pastorianus lager-brewing strains is therefore likely to have involved mechanisms similar to those observed in the present study.\n\nAuthor SummaryFermentation of the wort sugar maltotriose is critical for the flavor profile obtained during beer brewing. The recently discovered yeast Saccharomyces eubayanus is gaining popularity as an alternative to S. pastorianus and S. cerevisiae for brewing, however it is unable to utilize maltotriose. Here, a combination of non-GMO mutagenesis and laboratory evolution of the S. eubayanus type strain CBS 12357{top} was used to enable maltotriose fermentation in brewers wort. A resulting S. eubayanus strain showed a significantly improved brewing performance, including improved maltose and maltotriose consumption and a superior flavor profile. Whole genome sequencing identified a novel transporter gene, SeMALT413, which was formed by recombination between three different SeMALT maltose-transporter genes. Overexpression of SeMALT413 in CBS 12357{top} confirmed its neofunctionalization as a maltotriose transporter. The mosaic structure of the maltotriose transporter SpMty1 in S. pastorianus resembles that of SeMalt413, suggesting that maltotriose utilization likely emerged through similar recombination events during the domestication of current lager brewing strains.

evolutionary biology↗

bioRxiv2017

Nanopore sequencing enables near-complete de novo assembly of Saccharomyces cerevisiae reference strain CEN.PK113-7D

The haploid Saccharomyces cerevisiae strain CEN.PK113-7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C. However, these assemblies contain large sequence gaps, particularly in subtelomeric regions, and the assumption of perfect homology to S288C for scaffolding introduces bias.\n\nIn this study, we obtained a near-complete genome assembly of CEN.PK113-7D using only Oxford Nanopore Technologys MinION sequencing platform. 15 of the 16 chromosomes, the mitochondrial genome, and the 2-micron plasmid are assembled in single contigs and all but one chromosome starts or ends in a telomere cap. This improved genome assembly contains 770 Kbp of added sequence containing 248 gene annotations in comparison to the previous assembly of CEN.PK113-7D. Many of these genes encode functions determining fitness in specific growth conditions and are therefore highly relevant for various industrial applications. Furthermore, we discovered a translocation between chromosomes III and VIII which caused misidentification of a MAL locus in the previous CEN.PK113-7D assembly. This study demonstrates the power of long-read sequencing by providing a high-quality reference assembly and annotation of CEN.PK113-7D and places a caveat on assumed genome stability of microorganisms.

genomics↗

bioRxiv2017

A Di-Acidic Motif Targets Cytoplasmic Proteins For Unconventional Protein Secretion

We previously reported that Acb1, a cytoplasmic protein in Saccharomyces cerevisiae that cannot enter the endoplasmic reticulum (ER), was secreted upon nutrient starvation by a Vps4 independent, but ESCRT-I, -II and -III and Grh1 dependent pathway (Curwin et al., 2016). Here, we report that the same conditions result in secretion of another signal sequence lacking protein, superoxide dismutase 1 (SOD1). Similar to Acb1, SOD1 export requires Grh1 and a subset of ESCRT components. Importantly, our analysis reveals the existence of a conserved di-acidic motif (Asp-Glu) in SOD1 and Acb1 that is required for their respective export. This sequence is different from the di-acidic motif (Asp-X-Glu) necessary for export of transmembrane proteins from the ER. We propose that the Asp-Glu sequence acts as a targeting motif for the entry of SOD1 and Acb1, and likely many other proteins, upon nutrient starvation into a common albeit ER-Golgi independent pathway of secretion.

cell biology↗