STED super-resolution microscopy unveils the dynamics of Atg30 on yeast Pex3-labeled peroxisomes.

Peroxisomes are dynamic organelles with important metabolic functions. Yeast Pex3 is a multifunctional membrane protein aiding in peroxisomal biogenesis, inheritance, and degradation (pexophagy), by interacting with process-specific factors. Using multicolor (live-cell) stimulated emission depletion (STED) nanoscopy, we studied the localization of Pex3 and its binding partners in Unlike confocal microscopy, STED allows resolving the membrane of tiny peroxisomes, enabling accurate measurements of the size of all Pex3-labeled peroxisomes.

Overexpression of PEX14 results in mistargeting to mitochondria, accompanied by organelle fragmentation and clustering in human embryonic kidney cells.

Peroxisome biogenesis disorders are caused by pathogenic variants in genes involved in biogenesis and maintenance of peroxisomes. However, mitochondria are also often affected in these diseases. Peroxisomal membrane proteins, including PEX14, have been found to mislocalise to mitochondria in cells lacking peroxisomes.

The Hansenula polymorpha mitochondrial carrier family protein Mir1 is dually localized at peroxisomes and mitochondria.

Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified.

Hansenula polymorpha cells lacking the ER-localized peroxins Pex23 or Pex29 show defects in mitochondrial function and morphology.

Pex23 family proteins localize to the endoplasmic reticulum and play a role in peroxisome and lipid body formation. The yeast Hansenula polymorpha contains four members: Pex23, Pex24, Pex29 and Pex32. We previously showed that loss of Pex24 or Pex32 results in severe peroxisomal defects, caused by reduced peroxisome-endoplasmic reticulum contact sites.

Gluing yeast peroxisomes - composition and function of membrane contact sites.

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.

Novel targeting assay uncovers targeting information within peroxisomal ABC transporter Pxa1.

The mechanism behind peroxisomal membrane protein targeting is still poorly understood, with only two yeast proteins believed to be involved and no consensus targeting sequence. Pex19 is thought to bind peroxisomal membrane proteins in the cytosol, and is subsequently recruited by Pex3 at the peroxisomal surface, followed by protein insertion via a mechanism that is as-yet-unknown. However, some peroxisomal membrane proteins still correctly sort in the absence of Pex3 or Pex19, suggesting that multiple sorting pathways exist.

Correlative Light- and Electron Microscopy in Peroxisome Research.

Correlative light and electron microscopy (CLEM) combines the advantages of protein localization by fluorescence microscopy with the high resolution of electron microscopy. Here, we describe a protocol that we developed for yeast peroxisome research. First, cells are fixed, using conditions that preserve the properties of fluorescent proteins and avoid the introduction of autofluorescence.

Structure-function analysis of the ER-peroxisome contact site protein Pex32.

In the yeast , the ER protein Pex32 is required for associating peroxisomes to the ER. Here, we report on a structure-function analysis of Pex32. Localization studies of various Pex32 truncations showed that the N-terminal transmembrane domain of Pex32 is responsible for sorting.

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

In the yeast 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.

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

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 FF-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.

Comparative Genomics of Peroxisome Biogenesis Proteins: Making Sense of the PEX Proteins.

PEX genes encode proteins involved in peroxisome biogenesis and proliferation. Using a comparative genomics approach, we clarify the evolutionary relationships between the 37 known PEX proteins in a representative set of eukaryotes, including all common model organisms, pathogenic unicellular eukaryotes and human. A large number of previously unknown PEX orthologs were identified.

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

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.

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

The yeast contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four Pex23 family proteins localize to the endoplasmic reticulum (ER). Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome-ER contacts.

Flotillin-mediated membrane fluidity controls peptidoglycan synthesis and MreB movement.

The bacterial plasma membrane is an important cellular compartment. In recent years it has become obvious that protein complexes and lipids are not uniformly distributed within membranes. Current hypotheses suggest that flotillin proteins are required for the formation of complexes of membrane proteins including cell-wall synthetic proteins.

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

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.

Peroxisome Maintenance Depends on De Novo Peroxisome Formation in Yeast Mutants Defective in Peroxisome Fission and Inheritance.

There is an ongoing debate on how peroxisomes form: by growth and fission of pre-existing peroxisomes or de novo from another membrane. It has been proposed that, in wild type yeast cells, peroxisome fission and careful segregation of the organelles over mother cells and buds is essential for organelle maintenance. Using live cell imaging we observed that cells of the yeast , lacking the peroxisome fission protein Pex11, still show peroxisome fission and inheritance.

The peroxisome biogenesis factors Pex3 and Pex19: multitasking proteins with disputed functions.

The peroxisomal membrane protein (PMP) Pex3 and its cytosolic interaction partner Pex19 have been implicated in peroxisomal membrane biogenesis. Although these peroxins have been extensively studied, no consensus has been reached yet on how they operate. Here, we discuss two major models of their function, namely, in direct insertion of proteins into the peroxisomal membrane or in formation of PMP-containing vesicles from the endoplasmic reticulum (ER).

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.

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 β-oxidation of fatty acids and decomposition of HO, 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.

Assembly of the Mitochondrial Cristae Organizer Mic10 Is Regulated by Mic26-Mic27 Antagonism and Cardiolipin.

The multi-subunit mitochondrial contact site and cristae organizing system (MICOS) is a conserved protein complex of the inner mitochondrial membrane that is essential for maintenance of cristae architecture. The core subunit Mic10 forms large oligomers that build a scaffold and induce membrane curvature. The regulation of Mic10 oligomerization is poorly understood.

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

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.

Saccharomyces cerevisiae cells lacking Pex3 contain membrane vesicles that harbor a subset of peroxisomal membrane proteins.

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.

Impaired biosynthesis of the non-bilayer lipids phosphatidylethanolamine or cardiolipin does not affect peroxisome biogenesis and proliferation in Saccharomyces cerevisiae.

The non-bilayer forming lipids cardiolipin (CL) and phosphatidylethanolamine (PE) modulate membrane curvature, facilitate membrane fusion and affect the stability and function of membrane proteins. Yeast peroxisomal membranes contain significant amounts of CL and PE. We analysed the effect of CL deficiency and PE depletion on peroxisome biogenesis and proliferation in Saccharomyces cerevisiae.