68 research outputs found

    Yeast Vps13 is Crucial for Peroxisome Expansion in Cells With Reduced Peroxisome-ER Contact Sites

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    In the yeast Hansenula polymorpha the peroxisomal membrane protein Pex11 and three endoplasmic reticulum localized proteins of the Pex23 family (Pex23, Pex24 and Pex32) are involved in the formation of peroxisome-ER contact sites. Previous studies suggested that these contacts are involved in non-vesicular lipid transfer and important for expansion of the peroxisomal membrane. The absence of Pex32 results in a severe peroxisomal phenotype, while cells lacking Pex11, Pex23 or Pex24 show milder defects and still are capable to form peroxisomes and grow on methanol. We performed transposon mutagenesis on H. polymorpha pex11 cells and selected mutants that lost the capacity to grow on methanol and are severely blocked in peroxisome formation. This strategy resulted in the identification of Vps13, a highly conserved contact site protein involved in bulk lipid transfer. Our data show that peroxisome formation and function is normal in cells of a vps13 single deletion strain. However, Vps13 is essential for peroxisome biogenesis in pex11. Notably, Vps13 is also required for peroxisome formation in pex23 and pex24 cells. These data suggest that Vps13 is crucial for peroxisome formation in cells with reduced peroxisome-endoplasmic reticulum contact sites and plays a redundant function in lipid transfer from the ER to peroxisomes

    Iron regulates contrasting toxicity of uropathogenic <i>Escherichia coli</i> in macrophages and epithelial cells

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    By far most urinary tract infections are caused by genetically diverse uropathogenic Escherichia coli (UPEC). Knowledge of the virulence mechanisms of UPEC is critical for drug development, but most studies focus on only a single strain of UPEC. In this study, we compared the virulence mechanisms of four antibiotic-resistant and highly pathogenic UPEC isolates in human blood monocyte-derived macrophages and a bladder epithelial cell (BEC) line: ST999, ST131, ST1981 and ST95. We found that while non-pathogenic E. coli strains are efficiently killed by macrophages in bactericidal single membrane vacuoles, the UPEC strains survive within double-membrane vacuoles. On side-by-side comparison, we found that whereas ST999 only carries Fe3+ importers, ST95 carries both Fe2+ and Fe3+ importers and the toxins haemolysin and colibactin. Moreover, we found that ST999 grows in the Fe3+ rich vacuoles of BECs and macrophages with concomitant increased expression of haem receptor chuA and the hydrogen peroxide sensor oxyR. In contrast, ST95 produces toxins in iron-depleted conditions similar to that of the urinary tract. Whereas ST95 also persist in the iron rich vacuoles of BECs, it produces colibactin in response to low Fe3+ contributing to macrophage death. Thus, iron regulates the contrasting toxicities of UPEC strains in macrophages and bladder epithelial cells due to low and high labile iron concentrations, respectively

    Yeast cells contain a heterogeneous population of peroxisomes that segregate asymmetrically during cell division

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    Here we used fluorescence microscopy and a peroxisome-targeted tandem fluorescent protein timer to determine the relative age of peroxisomes in yeast. Our data indicate that yeast cells contain a heterogeneous population of relatively old and younger peroxisomes. During budding the peroxisome retention factor Inp1 selectively associates to the older organelles, which are retained in the mother cells. Inp2, a protein required for transport of peroxisomes to the bud, preferentially associates to younger organelles. Using a microfluidics device we demonstrate that the selective segregation of younger peroxisomes to the buds is carefully maintained during multiple budding events. The replicative lifespan of mother cells increased upon deletion of INP2, which resulted in the retention of all organelles in mother cells. These data suggest that in wild type yeast transport of aged and deteriorated peroxisomes to the bud is prevented, whereas the young and vital organelles are preferably transported to the newly forming buds

    Hansenula polymorpha Aat2p is targeted to peroxisomes via a novel Pex20p-dependent pathway

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    Saccharomyces cerevisiae Aat2p contains a peroxisomal targeting signal type-1 and localizes to peroxisomes in oleate-grown cells, but not in glucose-grown cells. Here, we have investigated Aat2p from the yeast Hansenula polymorpha, which lacks a recognizable peroxisomal targeting signal. Aat2p tagged with GFP at its C terminus displays a dual cytosol-peroxisome localization in ethanol-grown cells. The partial peroxisomal localization of Aat2p persisted in the absence of the classical cycling receptors Pex5p and Pex7p but Aat2p targeting to peroxisomes was reduced in cells deleted for the matrix protein import factors PEX1, PEX2 and PEX13. Furthermore, we demonstrate that Aat2p targeting to peroxisomes requires Pex20p. Together, our data identify a Pex20p-dependent pathway for targeting Aat2p to peroxisomes

    Peroxisome retention involves Inp1-dependent peroxisome-plasma membrane contact sites in yeast

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    ¬© 2020 Krikken et al.Retention of peroxisomes in yeast mother cells requires Inp1, which is recruited to the organelle by the peroxisomal membrane protein Pex3. Here we show that Hansenula polymorpha Inp1 associates peroxisomes to the plasma membrane. Peroxisome‚Äďplasma membrane contact sites disappear upon deletion of INP1 but increase upon INP1 overexpression. Analysis of truncated Inp1 variants showed that the C terminus is important for association to the peroxisome, while a stretch of conserved positive charges and a central pleckstrin homology-like domain are important for plasma membrane binding. In cells of a PEX3 deletion, strain Inp1-GFP localizes to the plasma membrane, concentrated in patches near the bud neck and in the cortex of nascent buds. Upon disruption of the actin cytoskeleton by treatment of the cells with latrunculin A, Inp1-GFP became cytosolic, indicating that Inp1 localization is dependent on the presence of an intact actin cytoskeleton.China Scholarship Council (NO AWARD)

    Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation in yeast

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    ¬© 2020. Published by The Company of Biologists Ltd.The yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome‚ÄďER contacts. Upon deletion of PEX24 or PEX32 ‚Äď and to a much lesser extent, of PEX23 or PEX29 ‚Äď peroxisome‚ÄďER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, and organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome‚ÄďER tether, indicating that Pex24 and Pex32 contribute to tethering of peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11, in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome‚ÄďER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.This work was supported by a grant from the FP7 People: Marie-Curie Actions Initial Training Networks (ITN) program PerFuMe (Grant Agreement Number 316723) to N.B., D.P.D. and I.J.v.d.K., from the China Scholarship Council (CSC) to F.W., and from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek/Chemical Sciences (NWO/CW) to A.A. (711.012.002)

    Hansenula polymorpha Pex37 is a peroxisomal membrane protein required for organelle fission and segregation

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    Here, we describe a novel peroxin, Pex37, in the yeast Hansenula polymorpha. H. polymorpha Pex37 is a peroxisomal membrane protein, which belongs to a protein family that includes, among others, the Neurospora crassa Woronin body protein Wsc, the human peroxisomal membrane protein PXMP2, the Saccharomyces cerevisiae mitochondrial inner membrane protein Sym1, and its mammalian homologue MPV17. We show that deletion of H. polymorpha PEX37 does not appear to have a significant effect on peroxisome biogenesis or proliferation in cells grown at peroxisome‚Äźinducing growth conditions (methanol). However, the absence of Pex37 results in a reduction in peroxisome numbers and a defect in peroxisome segregation in cells grown at peroxisome‚Äźrepressing conditions (glucose). Conversely, overproduction of Pex37 in glucose‚Äźgrown cells results in an increase in peroxisome numbers in conjunction with a decrease in their size. The increase in numbers in PEX37‚Äźoverexpressing cells depends on the dynamin‚Äźrelated protein Dnm1. Together our data suggest that Pex37 is involved in peroxisome fission in glucose‚Äźgrown cells. Introduction of human PXMP2 in H. polymorpha pex37 cells partially restored the peroxisomal phenotype, indicating that PXMP2 represents a functional homologue of Pex37. H.polymorpha pex37 cells did not show aberrant growth on any of the tested carbon and nitrogen sources that are metabolized by peroxisomal enzymes, suggesting that Pex37 may not fulfill an essential function in transport of these substrates or compounds required for their metabolism across the peroxisomal membrane.This work was supported by a grant from the Marie Curie Initial Training Networks (ITN) program PerFuMe (Grant Agreement Number 316723) to RS, NB, DPD, and IJvdK.Peer reviewe

    Yeast pex1 cells contain peroxisomal ghosts that import matrix proteins upon reintroduction of Pex1

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    Pex1 and Pex6 are two AAA-ATPases that play a crucial role in peroxisome biogenesis. We have characterized the ultrastructure of the Saccharomyces cerevisiae peroxisome-deficient mutants pex1 and pex6 by various high-resolution electron microscopy techniques. We observed that the cells contained peroxisomal membrane remnants, which in ultrathin cross sections generally appeared as double membrane rings. Electron tomography revealed that these structures consisted of one continuous membrane, representing an empty, flattened vesicle, which folds into a cup shape. Immunocytochemistry revealed that these structures lack peroxisomal matrix proteins but are the sole sites of the major peroxisomal membrane proteins Pex2, Pex10, Pex11, Pex13, and Pex14. Upon reintroduction of Pex1 in Pex1-deficient cells, these peroxisomal membrane remnants (ghosts) rapidly incorporated peroxisomal matrix proteins and developed into peroxisomes. Our data support earlier views that Pex1 and Pex6 play a role in peroxisomal matrix protein import

    Dual role of Mic10 in mitochondrial cristae organization and ATP synthase-linked metabolic adaptation and respiratory growth

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    Invaginations of the mitochondrial inner membrane, termed cristae, are hubs for oxidative phosphorylation. The mitochondrial contact site and cristae organizing system (MICOS) and the dimeric F(1)F(o)-ATP synthase play important roles in controlling cristae architecture. A fraction of the MICOS core subunit Mic10 is found in association with the ATP synthase, yet it is unknown whether this interaction is of relevance for mitochondrial or cellular functions. Here, we established conditions to selectively study the role of Mic10 at the ATP synthase. Mic10 variants impaired in MICOS functions stimulate ATP synthase oligomerization like wild-type Mic10 and promote efficient inner membrane energization, adaptation to non-fermentable carbon sources, and respiratory growth. Mic10's functions in respiratory growth largely depend on Mic10(ATPsynthase), not on Mic10(MICOS). We conclude that Mic10 plays a dual role as core subunit of MICOS and as partner of the F(1)F(o)-ATP synthase, serving distinct functions in cristae shaping and respiratory adaptation and growth

    Irregular particle morphology and membrane rupture facilitate ion gradients in the lumen of phagosomes

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    Localized fluxes, production, and/or degradation coupled to limited diffusion are well known to result in stable spatial concentration gradients of biomolecules in the cell. In this study, we demonstrate that this also holds true for small ions, since we found that the close membrane apposition between the membrane of a phagosome and the surface of the cargo particle it encloses, together with localized membrane rupture, suffice for stable gradients of protons and iron cations within the lumen of the phagosome. Our data show that, in phagosomes containing hexapod-shaped silica colloid particles, the phagosomal membrane is ruptured at the positions of the tips of the rods, but not at other positions. This results in the confined leakage at these positions of protons and iron from the lumen of the phagosome into the cytosol. In contrast, acidification and iron accumulation still occur at the positions of the phagosomes nearer to the cores of the particles. Our study strengthens the concept that coupling metabolic and signaling reaction cascades can be spatially confined by localized limited diffusion
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