scholarly journals Mutants of theYarrowia lipolytica PEX23Gene Encoding an Integral Peroxisomal Membrane Peroxin Mislocalize Matrix Proteins and Accumulate Vesicles Containing Peroxisomal Matrix and Membrane Proteins

2000 ◽  
Vol 11 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Trevor W. Brown ◽  
Vladimir I. Titorenko ◽  
Richard A. Rachubinski
Keyword(s):  
Membrane Proteins ◽  
Sequence Similarity ◽  
Integral Membrane Protein ◽  
Functional Complementation ◽  
Matrix Proteins ◽  
Hypothetical Proteins ◽  
Peroxisomal Membrane ◽  
High Sequence Similarity ◽  
Low Levels ◽  
Yeast Yarrowia Lipolytica

pex mutants are defective in peroxisome assembly. The mutant strain pex23-1 of the yeast Yarrowia lipolytica lacks morphologically recognizable peroxisomes and mislocalizes all peroxisomal matrix proteins investigated preferentially to the cytosol. pex23 strains accumulate vesicular structures containing both peroxisomal matrix and membrane proteins. The PEX23 gene was isolated by functional complementation of the pex23-1 strain and encodes a protein, Pex23p, of 418 amino acids (47,588 Da). Pex23p exhibits high sequence similarity to two hypothetical proteins of the yeastSaccharomyces cerevisiae. Pex23p is an integral membrane protein of peroxisomes that is completely, or nearly completely, sequestered from the cytosol. Pex23p is detected at low levels in cells grown in medium containing glucose, and its levels are significantly increased by growth in medium containing oleic acid, the metabolism of which requires intact peroxisomes.

scholarly journals Pex30p, Pex31p, and Pex32p Form a Family of Peroxisomal Integral Membrane Proteins Regulating Peroxisome Size and Number in Saccharomyces cerevisiae

2004 ◽  
Vol 15 (2) ◽  
pp. 665-677 ◽  
Author(s):  
Franco J. Vizeacoumar ◽  
Juan C. Torres-Guzman ◽  
David Bouard ◽  
John D. Aitchison ◽  
Richard A. Rachubinski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Yarrowia Lipolytica ◽  
Sequence Similarity ◽  
Integral Membrane Proteins ◽  
Peroxisome Proliferation ◽  
Hypothetical Proteins ◽  
Peroxisomal Membrane ◽  
High Sequence Similarity ◽  
Yeast Yarrowia Lipolytica

The peroxin Pex23p of the yeast Yarrowia lipolytica exhibits high sequence similarity to the hypothetical proteins Ylr324p, Ygr004p, and Ybr168p encoded by the Saccharomyces cerevisiae genome. Ylr324p, Ygr004p, and Ybr168p are integral to the peroxisomal membrane and act to control peroxisome number and size. Synthesis of Ylr324p and Ybr168p, but not of Ygr004p, is induced during incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for YLR324w exhibit increased numbers of peroxisomes, whereas cells deleted for YGR004w or YBR168w exhibit enlarged peroxisomes. Ylr324p and Ybr168p cannot functionally substitute for one another or for Ygr004p, whereas Ygr004p shows partial functional redundancy with Ylr324p and Ybr168p. Ylr324p, Ygr004p, and Ybr168p interact within themselves and with Pex28p and Pex29p, which have been shown also to regulate peroxisome size and number. Systematic deletion of genes demonstrated that PEX28 and PEX29 function upstream of YLR324w, YGR004w, and YBR168w in the regulation of peroxisome proliferation. Our data suggest a role for Ylr324p, Ygr004p, and Ybr168p—now designated Pex30p, Pex31p, and Pex32p, respectively—together with Pex28p and Pex29p in controlling peroxisome size and proliferation in Saccharomyces cerevisiae.

scholarly journals YHR150w and YDR479c encode peroxisomal integral membrane proteins involved in the regulation of peroxisome number, size, and distribution in Saccharomyces cerevisiae

2003 ◽  
Vol 161 (2) ◽  
pp. 321-332 ◽  
Author(s):  
Franco J. Vizeacoumar ◽  
Juan C. Torres-Guzman ◽  
Yuen Yi C. Tam ◽  
John D. Aitchison ◽  
Richard A. Rachubinski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Similarity ◽  
Buoyant Density ◽  
Integral Membrane Proteins ◽  
Hypothetical Proteins ◽  
Wild Type ◽  
Peroxisomal Membrane ◽  
High Sequence Similarity ◽  
Wild Type Phenotype ◽  
Yeast Yarrowia Lipolytica

The peroxin Pex24p of the yeast Yarrowia lipolytica exhibits high sequence similarity to two hypothetical proteins, Yhr150p and Ydr479p, encoded by the Saccharomyces cerevisiae genome. Like YlPex24p, both Yhr150p and Ydr479p have been shown to be integral to the peroxisomal membrane, but unlike YlPex24p, their levels of synthesis are not increased upon a shift of cells from glucose- to oleic acid–containing medium. Peroxisomes of cells deleted for either or both of the YHR150w and YDR479c genes are increased in number, exhibit extensive clustering, are smaller in area than peroxisomes of wild-type cells, and often exhibit membrane thickening between adjacent peroxisomes in a cluster. Peroxisomes isolated from cells deleted for both genes have a decreased buoyant density compared with peroxisomes isolated from wild-type cells and still exhibit clustering and peroxisomal membrane thickening. Overexpression of the genes PEX25 or VPS1, but not the gene PEX11, restored the wild-type phenotype to cells deleted for one or both of the YHR150w and YDR479c genes. Together, our data suggest a role for Yhr150p and Ydr479p, together with Pex25p and Vps1p, in regulating peroxisome number, size, and distribution in S. cerevisiae. Because of their role in peroxisome dynamics, YHR150w and YDR479c have been designated as PEX28 and PEX29, respectively, and their encoded peroxins as Pex28p and Pex29p.

scholarly journals Yarrowia lipolyticaCells Mutant for the Peroxisomal Peroxin Pex19p Contain Structures Resembling Wild-Type Peroxisomes

2001 ◽  
Vol 12 (11) ◽  
pp. 3353-3364 ◽  
Author(s):  
Gareth R. Lambkin ◽  
Richard A. Rachubinski
Keyword(s):  
Oleic Acid ◽  
Membrane Protein ◽  
Gradient Centrifugation ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Matrix Proteins ◽  
Wild Type ◽  
Peroxisomal Membrane ◽  
Protein Receptor ◽  
Yeast Yarrowia Lipolytica

PEX genes encode peroxins, which are proteins required for peroxisome assembly. The PEX19 gene of the yeast Yarrowia lipolytica was isolated by functional complementation of the oleic acid-nonutilizing strainpex19-1 and encodes Pex19p, a protein of 324 amino acids (34,822 Da). Subcellular fractionation and immunofluorescence microscopy showed Pex19p to be localized primarily to peroxisomes. Pex19p is detected in cells grown in glucose-containing medium, and its levels are not increased by incubation of cells in oleic acid–containing medium, the metabolism of which requires intact peroxisomes. pex19 cells preferentially mislocalize peroxisomal matrix proteins and the peripheral intraperoxisomal membrane peroxin Pex16p to the cytosol, although small amounts of these proteins could be reproducibly localized to a subcellular fraction enriched for peroxisomes. In contrast, the peroxisomal integral membrane protein Pex2p exhibits greatly reduced levels inpex19 cells compared with its levels in wild-type cells. Importantly, pex19 cells were shown by electron microscopy to contain structures that resemble wild-type peroxisomes in regards to size, shape, number, and electron density. Subcellular fractionation and isopycnic density gradient centrifugation confirmed the presence of vesicular structures in pex19 mutant strains that were similar in density to wild-type peroxisomes and that contained profiles of peroxisomal matrix and membrane proteins that are similar to, yet distinct from, those of wild-type peroxisomes. Because peroxisomal structures form in pex19 cells, Pex19p apparently does not function as a peroxisomal membrane protein receptor in Y. lipolytica. Our results are consistent with a role for Y. lipolytica Pex19p in stabilizing the peroxisomal membrane.

scholarly journals The peroxin Pex17p of the yeast Yarrowia lipolytica is associated peripherally with the peroxisomal membrane and is required for the import of a subset of matrix proteins.

1997 ◽  
Vol 17 (5) ◽  
pp. 2511-2520 ◽  
Author(s):  
J J Smith ◽  
R K Szilard ◽  
M Marelli ◽  
R A Rachubinski
Keyword(s):  
Amino Acids ◽  
Oleic Acid ◽  
Yarrowia Lipolytica ◽  
Matrix Proteins ◽  
Peroxisomal Membrane ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Carboxyl Terminal ◽  
Peroxisomal Targeting Signal ◽  
Yeast Yarrowia Lipolytica

PEX genes encode peroxins, which are required for the biogenesis of peroxisomes. The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da. Pex17p is peripherally associated with the peroxisomal membrane. The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes. Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium. Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures. The import of peroxisomal matrix proteins in these mutants is selectively impaired. This selective import is not a function of the nature of the peroxisomal targeting signal. We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.

scholarly journals Multiple Distinct Targeting Signals in Integral Peroxisomal Membrane Proteins

2001 ◽  
Vol 153 (6) ◽  
pp. 1141-1150 ◽  
Author(s):  
Jacob M. Jones ◽  
James C. Morrell ◽  
Stephen J. Gould
Keyword(s):  
Membrane Proteins ◽  
Matrix Proteins ◽  
Membrane Biogenesis ◽  
Peroxisomal Membrane ◽  
Interesting Possibility ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Peroxisome Membrane ◽  
Metabolite Transporter

Peroxisomal proteins are synthesized on free polysomes and then transported from the cytoplasm to peroxisomes. This process is mediated by two short well-defined targeting signals in peroxisomal matrix proteins, but a well-defined targeting signal has not yet been described for peroxisomal membrane proteins (PMPs). One assumption in virtually all prior studies of PMP targeting is that a given protein contains one, and only one, distinct targeting signal. Here, we show that the metabolite transporter PMP34, an integral PMP, contains at least two nonoverlapping sets of targeting information, either of which is sufficient for insertion into the peroxisome membrane. We also show that another integral PMP, the peroxin PEX13, also contains two independent sets of peroxisomal targeting information. These results challenge a major assumption of most PMP targeting studies. In addition, we demonstrate that PEX19, a factor required for peroxisomal membrane biogenesis, interacts with the two minimal targeting regions of PMP34. Together, these results raise the interesting possibility that PMP import may require novel mechanisms to ensure the solubility of integral PMPs before their insertion in the peroxisome membrane, and that PEX19 may play a central role in this process.

scholarly journals Mutants of the Yeast Yarrowia lipolyticaDefective in Protein Exit from the Endoplasmic Reticulum Are Also Defective in Peroxisome Biogenesis

1998 ◽  
Vol 18 (5) ◽  
pp. 2789-2803 ◽  
Author(s):  
Vladimir I. Titorenko ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Oleic Acid ◽  
Membrane Proteins ◽  
Matrix Proteins ◽  
Secretory Proteins ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Mutant Cells

ABSTRACT Mutations in the SEC238 and SRP54 genes of the yeast Yarrowia lipolytica not only cause temperature-sensitive defects in the exit of the precursor form of alkaline extracellular protease and of other secretory proteins from the endoplasmic reticulum and in protein secretion but also lead to temperature-sensitive growth in oleic acid-containing medium, the metabolism of which requires the assembly of functionally intact peroxisomes. The sec238A andsrp54KO mutations at the restrictive temperature significantly reduce the size and number of peroxisomes, affect the import of peroxisomal matrix and membrane proteins into the organelle, and significantly delay, but do not prevent, the exit of two peroxisomal membrane proteins, Pex2p and Pex16p, from the endoplasmic reticulum en route to the peroxisomal membrane. Mutations in the PEX1 and PEX6 genes, which encode members of the AAA family of N-ethylmaleimide-sensitive fusion protein-like ATPases, not only affect the exit of precursor forms of secretory proteins from the endoplasmic reticulum but also prevent the exit of the peroxisomal membrane proteins Pex2p and Pex16p from the endoplasmic reticulum and cause the accumulation of an extensive network of endoplasmic reticulum membranes. None of the peroxisomal matrix proteins tested associated with the endoplasmic reticulum in sec238A,srp54KO, pex1-1, and pex6KO mutant cells. Our data provide evidence that the endoplasmic reticulum is required for peroxisome biogenesis and suggest that inY. lipolytica, the trafficking of some membrane proteins, but not matrix proteins, to the peroxisome occurs via the endoplasmic reticulum, results in their glycosylation within the lumen of the endoplasmic reticulum, does not involve transport through the Golgi, and requires the products encoded by the SEC238, SRP54,PEX1, and PEX6 genes.

scholarly journals Design, purification and characterization of a soluble variant of the integral membrane protein MotB for structural studies

2013 ◽  
Vol 10 (79) ◽  
pp. 20120717 ◽  
Author(s):  
Daniel A. Andrews ◽  
Meng Xie ◽  
Victoria Hughes ◽  
Matthew C. Wilce ◽  
Anna Roujeinikova
Keyword(s):  
Membrane Proteins ◽  
Leucine Zipper ◽  
Integral Membrane Protein ◽  
Flagellar Motor ◽  
Structural Studies ◽  
Transmembrane Helices ◽  
Biophysical Analysis ◽  
Torque Generation ◽  
Low Levels

The bacterial flagellar motor is an intricate nanomachine powered by a transmembrane electrochemical gradient. Rotation is driven by the cumulative action of several peptidoglycan-anchored stator complexes on the rotor. In proton-motive force-driven motors, the stator complex is composed of a motility protein B (MotB) dimer surrounded by four copies of MotA, where both MotA and MotB are integral membrane proteins. The lack of full-length MotA and MotB structures hinders understanding of the mechanism of torque generation. Given the low levels of expression and low stability of detergent-solubilized MotB, a soluble chimaeric variant was engineered, where the two transmembrane helices of the MotB dimer were replaced by a leucine zipper. The biochemical and biophysical analysis of the resultant protein showed that it was properly folded, stable, behaved as a monodisperse dimer at low pH, had molecular dimensions close to those expected for native MotB and yielded reproducible crystals. The chimaeric protein is, therefore, a good candidate for structural studies. This ‘solubilization by design’ approach may be generally applicable to the production of soluble forms of other dimeric, trimeric and tetrameric single-span membrane proteins for functional and structural studies.

scholarly journals Peroxisome Synthesis in the Absence of Preexisting Peroxisomes

1999 ◽  
Vol 144 (2) ◽  
pp. 255-266 ◽  
Author(s):  
Sarah T. South ◽  
Stephen J. Gould
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Matrix Protein ◽  
Protein Import ◽  
Zellweger Syndrome ◽  
Matrix Proteins ◽  
Peroxisomal Membrane ◽  
Peroxisomal Membrane Protein ◽  
The Matrix ◽  
Inactivating Mutations

Zellweger syndrome and related diseases are caused by defective import of peroxisomal matrix proteins. In all previously reported Zellweger syndrome cell lines the defect could be assigned to the matrix protein import pathway since peroxisome membranes were present, and import of integral peroxisomal membrane proteins was normal. However, we report here a Zellweger syndrome patient (PBD061) with an unusual cellular phenotype, an inability to import peroxisomal membrane proteins. We also identified human PEX16, a novel integral peroxisomal membrane protein, and found that PBD061 had inactivating mutations in the PEX16 gene. Previous studies have suggested that peroxisomes arise from preexisting peroxisomes but we find that expression of PEX16 restores the formation of new peroxisomes in PBD061 cells. Peroxisome synthesis and peroxisomal membrane protein import could be detected within 2–3 h of PEX16 injection and was followed by matrix protein import. These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes. We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes.

scholarly journals Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 27 ◽  
Author(s):  
Claudia Colasante ◽  
Frank Voncken ◽  
Theresa Manful ◽  
Thomas Ruppert ◽  
Aloysius G M Tielens ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Trypanosoma Brucei ◽  
Metabolic Pathways ◽  
Matrix Proteins ◽  
Membrane Domains ◽  
Purine Salvage ◽  
Peroxisomal Membrane ◽  
Leishmania Tarentolae ◽  
Mitochondrial Carrier ◽  
Mitochondrial Carrier Protein

In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

scholarly journals Pex11-related Proteins in Peroxisome Dynamics: A Role for the Novel Peroxin Pex27p in Controlling Peroxisome Size and Number in Saccharomyces cerevisiae

2003 ◽  
Vol 14 (10) ◽  
pp. 4089-4102 ◽  
Author(s):  
Yuen Yi C. Tam ◽  
Juan C. Torres-Guzman ◽  
Franco J. Vizeacoumar ◽  
Jennifer J. Smith ◽  
Marcello Marelli ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Sequence Similarity ◽  
Transcriptome Profiling ◽  
The Novel ◽  
Yeast Two Hybrid ◽  
Peroxisomal Membrane ◽  
High Sequence Similarity ◽  
Reading Frame ◽  
Related Proteins ◽  
Two Hybrid

Transcriptome profiling identified the gene PEX25 encoding Pex25p, a peroxisomal membrane peroxin required for the regulation of peroxisome size and maintenance in Saccharomyces cerevisiae. Pex25p is related to a protein of unknown function encoded by the open reading frame, YOR193w, of the S. cerevisiae genome. Yor193p is a peripheral peroxisomal membrane protein that exhibits high sequence similarity not only to Pex25p but also to the peroxisomal membrane peroxin Pex11p. Unlike Pex25p and Pex11p, Yor193p is constitutively expressed in wild-type cells grown in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for the YOR193w gene show a few enlarged peroxisomes. Peroxisomes are greatly enlarged in cells harboring double deletions of the YOR193w and PEX25 genes, the YOR193w and PEX11 genes, and the PEX25 and PEX11 genes. Yeast two-hybrid analyses showed that Yor193p interacts with Pex25p and itself, Pex25p interacts with Yor193p and itself, and Pex11p interacts only with itself. Overexpression of YOR193w, PEX25, or PEX11 led to peroxisome proliferation and the formation of small peroxisomes. Our data suggest a role for Yor193p, renamed Pex27p, in controlling peroxisome size and number in S. cerevisiae.

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