Search

Membrane contact sites are defined as regions of close proximity between two membranes; this association is mediated by protein-protein and/or protein-lipid interactions. Contact sites are often involved in lipid transport, but also can perform other functions. Peroxisomal membrane contact sites have obtained little attention compared to those of other cell organelles. However, recent studies resulted in a big leap in our knowledge of the occurrence, composition and function of peroxisomal contact sites. Studies in yeast strongly contributed to this progress. In this Review, we present an overview of our current knowledge on peroxisomal membrane contact sites in various yeast species, including Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica. Yeast peroxisomes form contacts with almost all other cellular organelles and with the plasma membrane. The absence of a component of a yeast peroxisomal contact site complex results in a range of peroxisomal phenotypes, including metabolic and biogenesis defects and alterations in organelle number, size or position. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae Vac8 is a vacuolar membrane protein, which functions in vacuole inheritance and fusion, nucleus-vacuole junctions, autophagy and the cytoplasm-to-vacuole-targeting pathway. Here, we analyzed Vac8 of the yeast Hansenula polymorpha. We show that Hp Vac8 localizes to the vacuolar membrane and concentrates in patches at nucleus-vacuole junctions. Analysis of a VAC8 deletion strain indicated that Hp Vac8 is required for vacuole inheritance and the formation of nuclear-vacuole junctions, but not for vacuole fusion. Previously, organelle proteomics resulted in the identification of Vac8 in peroxisomal fractions isolated from H. polymorpha and S. cerevisiae. However, deletion of H. polymorpha VAC8 had no effect on peroxisome biogenesis or peroxisome-vacuole contact sites. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlights • Yeast peroxisomal membranes form contacts with several other membranes. • At peroxisome inducing conditions peroxisome-vacuole contacts are formed. • Pex3 accumulates in patches at peroxisome-vacuole contacts. • Pex3 overproduction results in the formation of peroxisome-vacuole contacts. [ABSTRACT FROM AUTHOR]

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 young peroxisomes. During budding, the peroxisome retention factor inheritance of peroxisomes protein 1 (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. [ABSTRACT FROM AUTHOR]

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--plasma 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. [ABSTRACT FROM AUTHOR]

Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner. [ABSTRACT FROM AUTHOR]