The dynamin-related protein Vps1 and the peroxisomal membrane protein Pex27 function together during peroxisome fission

Dynamin-related proteins (Drps) mediate a variety of membrane remodelling processes. The Saccharomyces cerevisiae Drp, Vps1, is required for endocytosis, endosomal sorting, vacuole fusion, and peroxisome fission and breakdown. How Drps, and in particular Vps1, can function at so many different subcellular locations is of interest to our understanding of cellular organisation. We found that the peroxisomal membrane protein Pex27 is specifically required for Vps1-dependent peroxisome fission in proliferating cells but is not required for Dnm1-dependent peroxisome fission. Pex27 accumulates in constricted regions of peroxisomes and affects peroxisome geometry upon overexpression. Moreover, Pex27 physically interacts with Vps1 in vivo and is required for the accumulation of a GTPase-defective Vps1 mutant (K42A) on peroxisomes. During nitrogen starvation, a condition that halts cell division and induces peroxisome breakdown, Vps1 associates with the pexophagophore. Pex27 is neither required for Vps1 recruitment to the pexophagophore nor for pexophagy. Our study identifies Pex27 as a Vps1-specific partner for the maintenance of peroxisome number in proliferating yeast cells

Pnc1 piggy-back import into peroxisomes relies on Gpd1 homodimerisation.

Peroxisomes are eukaryotic organelles that posttranslationally import proteins via one of two conserved peroxisomal targeting signal (PTS1 or 2) mediated pathways. Oligomeric proteins can be imported via these pathways but evidence is accumulating that at least some PTS1-containing monomers enter peroxisomes before they assemble into oligomers. Some proteins lacking a PTS are imported by piggy-backing onto PTS-containing proteins. One of these proteins is the nicotinamidase Pnc1, that is co-imported with the PTS2-containing enzyme Glycerol-3-phosphate dehydrogenase 1, Gpd1. Here we show that Pnc1 co-import requires Gpd1 to form homodimers. A mutation that interferes with Gpd1 homodimerisation does not prevent Gpd1 import but prevents Pnc1 co-import. A suppressor mutation that restores Gpd1 homodimerisation also restores Pnc1 co-import. In line with this, Pnc1 interacts with Gpd1 in vivo only when Gpd1 can form dimers. Redirection of Gpd1 from the PTS2 import pathway to the PTS1 import pathway supports Gpd1 monomer import but not Gpd1 homodimer import and Pnc1 co-import. Our results support a model whereby Gpd1 may be imported as a monomer or a dimer but only the Gpd1 dimer facilitates co-transport of Pnc1 into peroxisomes

Insights into the regulation of the mitochondrial inheritance and trafficking adaptor protein Mmr1 in saccharomyces cerevisiae

Mitochondria are organelles involved in cellular energetics in all eukaryotes, and changes in their dynamics, fission, fusion, or localization can lead to cell defects and disease in humans. Budding yeast, Saccharomyces cerevisiae, has been shown to be an effective model organism in elucidating mechanisms underpinning these mitochondrial processes. In the work presented here, a genetic screen was performed to identify overexpressing kinases, phosphatases, and ubiquitin ligases, which resulted in mitochondrial defects. A total of 33 overexpressed genes showed mitochondrial phenotypes but without severe growth defects. These included a subset that affected the timing of mitochondrial inheritance and were the focus of further study. Using cell and biochemical approaches, the roles of the PAK-family kinase Cla4 and the E3-ubiquitin ligases Dma1 and Dma2 were investigated. Previous studies have indicated the roles of kinase Cla4 and ligases Dma1 and Dma2 in triggering the degradation of trafficking adaptors in the bud, which leads to disruption of the interaction with the transporting class V myosin, Myo2. Here, we map a key interface between Cla4 and the mitochondrial adaptor Mmr1 necessary for phosphorylation and identify a region of Mmr1 required for its degradation via Dma1 and Dma2. Together, our data provide insights into key regulatory regions of Mmr1 responsible for its function in mitochondrial inheritance

The Pex3–Inp1 complex tethers yeast peroxisomes to the plasma membrane

A subset of peroxisomes is retained at the mother cell cortex by the Pex3–Inp1 complex. We identify Inp1 as the first known plasma membrane–peroxisome (PM-PER) tether by demonstrating that Inp1 meets the predefined criteria that a contact site tether protein must adhere to. We show that Inp1 is present in the correct subcellular location to interact with both the plasma membrane and peroxisomal membrane and has the structural and functional capacity to be a PM-PER tether. Additionally, expression of artificial PM-PER tethers is sufficient to restore retention in inp1Δ cells. We show that Inp1 mediates peroxisome retention via an N-terminal domain that binds PI(4,5)P2 and a C-terminal Pex3-binding domain, forming a bridge between the peroxisomal membrane and the plasma membrane. We provide the first molecular characterization of the PM-PER tether and show it anchors peroxisomes at the mother cell cortex, suggesting a new model for peroxisome retention

Variation in gene copy number and polymorphism of the human salivary amylase isoenzyme system in Caucasians

The polymorphic patterns of human salivary amylase of a large number of individuals of Caucasian origin were determined by using isoelectric focusing and polyacrylamide gel electrophoresis. Nine different salivary amylase protein variants were found; three of them are recorded for the first time and their heredity is shown. Some of the variants are encoded by haplotypes expressing three allozymes. Most variants display low frequencies. Analysis of the relative intensities of variant-specific isozyme bands, combined with segregation analysis, show that extensive quantitative variation is present in the population. The numbers of salivary amylase genes in some families showing quantitative variation at the protein level have been estimated by the polymerase chain reaction. We present evidence that quantitative variations in amylase protein patterns do not always reflect variations in gene copy number but that other mechanisms are also involved. © 1992 Springer-Verlag

Proteasome-dependent protein quality control of the peroxisomal membrane protein Pxa1p

Peroxisomes are eukaryotic organelles that function in numerous metabolic pathways and defects in peroxisome function can cause serious developmental brain disorders such as adrenoleukodystrophy (ALD). Peroxisomal membrane proteins (PMPs) play a crucial role in regulating peroxisome function. Therefore, PMP homeostasis is vital for peroxisome function. Recently, we established that certain PMPs are degraded by the Ubiquitin Proteasome System yet little is known about how faulty/non-functional PMPs undergo quality control. Here we have investigated the degradation of Pxa1p, a fatty acid transporter in the yeast Saccharomyces cerevisiae. Pxa1p is a homologue of the human protein ALDP and mutations in ALDP result in the severe disorder ALD. By introducing two corresponding ALDP mutations into Pxa1p (Pxa1MUT), fused to mGFP, we show that Pxa1MUT-mGFP is rapidly degraded from peroxisomes in a proteasome-dependent manner, while wild type Pxa1-mGFP remains relatively stable. Furthermore, we identify a role for the ubiquitin ligase Ufd4p in Pxa1MUT-mGFP degradation. Finally, we establish that inhibiting Pxa1MUT-mGFP degradation results in a partial rescue of Pxa1p activity in cells. Together, our data demonstrate that faulty PMPs can undergo proteasome-dependent quality control. Furthermore, our observations may provide new insights into the role of ALDP degradation in ALD

Intra-ER sorting of the peroxisomal membrane protein Pex3 relies on its luminal domain.

Pex3 is an evolutionarily conserved type III peroxisomal membrane protein required for peroxisome formation. It is inserted into the ER membrane and sorted via an ER subdomain (the peroxisomal ER, or pER) to peroxisomes. By constructing chimeras between Pex3 and the type III ER membrane protein Sec66, we have been able to separate the signals that mediate insertion of Pex3 into the ER from those that mediate sorting within the ER to the pER subdomain. The N-terminal 17-amino acid segment of Pex3 contains two signals that are each sufficient for sorting to the pER: a chimeric protein containing the N-terminal domain of Pex3 fused to the transmembrane and cytoplasmic segments of Sec66 sorts to the pER in wild type cells, and does not colocalise with peroxisomes. Subsequent transport to existing peroxisomes requires the Pex3 transmembrane segment. When expressed in Drosophila S2R+ cells, ScPex3 targeting to peroxisomes is dependent on the intra-ER sorting signals in the N-terminal segment. The N-terminal segments of both human and Drosophila Pex3 contain intra-ER sorting information and can replace that of ScPex3. Our analysis has uncovered the signals within Pex3 required for the various steps of its transport to peroxisomes. Our generation of versions of Pex3 that are blocked at each stage along its transport pathway provides a tool to dissect the mechanism, as well as the molecular machinery required at each step of the pathway

Spindle position checkpoint kinase Kin4 regulates organelle transport in Saccharomyces cerevisiae

Membrane-bound organelles play important, frequently essential, roles in cellular metabolism in eukaryotes. Hence, cells have evolved molecular mechanisms to closely monitor organelle dynamics and maintenance. The actin cytoskeleton plays a vital role in organelle transport and positioning across all eukaryotes. Studies in the budding yeast Saccharomyces cerevisiae (S. cerevisiae) revealed that a block in actomyosin-dependent transport affects organelle inheritance to daughter cells. Indeed, class V Myosins, Myo2, and Myo4, and many of their organelle receptors, have been identified as key factors in organelle inheritance. However, the spatiotemporal regulation of yeast organelle transport remains poorly understood. Using peroxisome inheritance as a proxy to study actomyosin-based organelle transport, we performed an automated genome-wide genetic screen in S. cerevisiae. We report that the spindle position checkpoint (SPOC) kinase Kin4 and, to a lesser extent, its paralog Frk1, regulates peroxisome transport, independent of their role in the SPOC. We show that Kin4 requires its kinase activity to function and that both Kin4 and Frk1 protect Inp2, the peroxisomal Myo2 receptor, from degradation in mother cells. In addition, vacuole inheritance is also affected in kin4/frk1-deficient cells, suggesting a common regulatory mechanism for actin-based transport for these two organelles in yeast. More broadly our findings have implications for understanding actomyosin-based transport in cells

Efficient PCR-based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology

Peroxisomal NAD(H) homeostasis in the yeast debaryomyces hansenii depends on two redox shuttles and the NAD+ carrier, Pmp47

Debaryomyces hansenii is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid β-oxidation pathway in D. hansenii. To this end, we employed recently developed methods to generate multiple gene deletions and tag open reading frames with GFP in their chromosomal context in this yeast. We found that, similar as in other yeasts, the β-oxidation of fatty acids in D. hansenii was restricted to peroxisomes. We report a series of experiments in D. hansenii and the well-studied yeast Saccharomyces cerevisiae that show that the homeostasis of NAD+ in D. hansenii peroxisomes is dependent upon the peroxisomal membrane protein Pmp47 and two peroxisomal dehydrogenases, Mdh3 and Gpd1, which both export reducing equivalents produced during β-oxidation to the cytosol. Pmp47 is the first identified NAD+ carrier in yeast peroxisomes