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

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.

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

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-ER contacts. Upon deletion of PEX24 or PEX32 - and to a much lesser extent, of PEX23 or PEX29 - peroxisome-ER 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-ER 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-ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 deletion cells.

Age-dependent deterioration of nuclear pore assembly in mitotic cells decreases transport dynamics.

Nuclear transport is facilitated by the Nuclear Pore Complex (NPC) and is essential for life in eukaryotes. The NPC is a long-lived and exceptionally large structure. We asked whether NPC quality control is compromised in aging mitotic cells. Our images of single yeast cells during aging, show that the abundance of several NPC components and NPC assembly factors decreases. Additionally, the single-cell life histories reveal that cells that better maintain those components are longer lived. The presence of herniations at the nuclear envelope of aged cells suggests that misassembled NPCs are accumulated in aged cells. Aged cells show decreased dynamics of transcription factor shuttling and increased nuclear compartmentalization. These functional changes are likely caused by the presence of misassembled NPCs, as we find that two NPC assembly mutants show similar transport phenotypes as aged cells. We conclude that NPC interphase assembly is a major challenge for aging mitotic cells.

Peroxisome development in yeast is associated with the formation of Pex3-dependent peroxisome-vacuole contact sites.

Using electron and fluorescence microscopy techniques, we identified various physical contacts between peroxisomes and other cell organelles in the yeast Hansenula polymorpha. In exponential glucose-grown cells, which typically contain a single small peroxisome, contacts were only observed with the endoplasmic reticulum and the plasma membrane. Here we focus on a novel peroxisome-vacuole contact site that is formed when glucose-grown cells are shifted to methanol containing media, conditions that induce strong peroxisome development. At these conditions, the small peroxisomes rapidly increase in size, a phenomenon that is paralleled by the formation of distinct intimate contacts with the vacuole. Localization studies showed that the peroxin Pex3 accumulated in patches at the peroxisome-vacuole contact sites. In wild-type cells growing exponentially on medium containing glucose, peroxisome-vacuole contact sites were never observed. However, upon overproduction of Pex3 peroxisomes also associated to vacuoles at these growth conditions. Our observations strongly suggest a role for Pex3 in the formation of a novel peroxisome-vacuole contact site. This contact likely plays a role in membrane growth as it is formed solely at conditions of strong peroxisome expansion.

Yeast peroxisomes: How are they formed and how do they grow?

Peroxisomes are single membrane enclosed cell organelles, which are present in almost all eukaryotic cells. In addition to the common peroxisomal pathways such as ß-oxidation of fatty acids and decomposition of H2O2, these organelles fulfil a range of metabolic and non-metabolic functions. Peroxisomes are very important since various human disorders exist that are caused by a defect in peroxisome function. Here we describe our current knowledge on the molecular mechanisms of peroxisome biogenesis in yeast, including peroxisomal protein sorting, organelle dynamics and peroxisomal membrane contact sites.

In vivo evolutionary engineering for ethanol-tolerance of Saccharomyces cerevisiae haploid cells triggers diploidization.

Microbial ethanol production is an important alternative energy resource to replace fossil fuels, but at high level, this product is highly toxic, which hampers its efficient production. Towards increasing ethanol-tolerance of Saccharomyces cerevisiae, the so far best industrial ethanol-producer, we evaluated an in vivo evolutionary engineering strategy based on batch selection under both constant (5%, v v-1) and gradually increasing (5-11.4%, v v-1) ethanol concentrations. Selection under increasing ethanol levels yielded evolved clones that could tolerate up to 12% (v v-1) ethanol and had cross-resistance to other stresses. Quite surprisingly, diploidization of the yeast population took place already at 7% (v v-1) ethanol level during evolutionary engineering, and this event was abolished by the loss of MKT1, a gene previously identified as being implicated in ethanol tolerance (Swinnen et al., Genome Res., 22, 975-984, 2012). Transcriptomic analysis confirmed diploidization of the evolved clones with strong down-regulation in mating process, and in several haploid-specific genes. We selected two clones exhibiting the highest viability on 12% ethanol, and found productivity and titer of ethanol significantly higher than those of the reference strain under aerated fed-batch cultivation conditions. This higher fermentation performance could be related with a higher abundance of glycolytic and ribosomal proteins and with a relatively lower respiratory capacity of the evolved strain, as revealed by a comparative transcriptomic and proteomic analysis between the evolved and the reference strains. Altogether, these results emphasize the efficiency of the in vivo evolutionary engineering strategy for improving ethanol tolerance, and the link between ethanol tolerance and diploidization.