scholarly journals Inhibitors of Copi and Copii Do Not Block PEX3-Mediated Peroxisome Synthesis

2000 ◽  
Vol 149 (7) ◽  
pp. 1345-1360 ◽  
Author(s):  
Sarah T. South ◽  
Katherine A. Sacksteder ◽  
Xiaoling Li ◽  
Yifei Liu ◽  
Stephen J. Gould
Keyword(s):  
Membrane Proteins ◽  
Zellweger Syndrome ◽  
Brefeldin A ◽  
Membrane Traffic ◽  
Dominant Negative ◽  
Peroxisome Biogenesis ◽  
Membrane Synthesis ◽  
Peroxisomal Membrane ◽  
Peroxisome Membrane ◽  
Inactivating Mutations

In humans, defects in peroxisome biogenesis are the cause of lethal diseases typified by Zellweger syndrome. Here, we show that inactivating mutations in human PEX3 cause Zellweger syndrome, abrogate peroxisome membrane synthesis, and result in reduced abundance of peroxisomal membrane proteins (PMPs) and/or mislocalization of PMPs to the mitochondria. Previous studies have suggested that PEX3 may traffic through the ER en route to the peroxisome, that the COPI inhibitor, brefeldin A, leads to accumulation of PEX3 in the ER, and that PEX3 overexpression alters the morphology of the ER. However, we were unable to detect PEX3 in the ER at early times after expression. Furthermore, we find that inhibition of COPI function by brefeldin A has no effect on trafficking of PEX3 to peroxisomes and does not inhibit PEX3-mediated peroxisome biogenesis. We also find that inhibition of COPII-dependent membrane traffic by a dominant negative SAR1 mutant fails to block PEX3 transport to peroxisomes and PEX3-mediated peroxisome synthesis. Based on these results, we propose that PEX3 targeting to peroxisomes and PEX3-mediated peroxisome membrane synthesis may occur independently of COPI- and COPII-dependent membrane traffic.

scholarly journals The Peroxin Pex19p Interacts with Multiple, Integral Membrane Proteins at the Peroxisomal Membrane

2000 ◽  
Vol 149 (6) ◽  
pp. 1171-1178 ◽  
Author(s):  
William B. Snyder ◽  
Antonius Koller ◽  
A. Jobu Choy ◽  
Suresh Subramani
Keyword(s):  
Membrane Proteins ◽  
Multiple Integral ◽  
Integral Membrane Proteins ◽  
Peroxisome Biogenesis ◽  
Yeast Two Hybrid ◽  
Peroxisomal Membrane ◽  
Site Of Action ◽  
Peroxisome Membrane ◽  
Two Hybrid ◽  
New Protein

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome.

scholarly journals Pex19 Binds Multiple Peroxisomal Membrane Proteins, Is Predominantly Cytoplasmic, and Is Required for Peroxisome Membrane Synthesis

2000 ◽  
Vol 148 (5) ◽  
pp. 931-944 ◽  
Author(s):  
Katherine A. Sacksteder ◽  
Jacob M. Jones ◽  
Sarah T. South ◽  
Xiaoling Li ◽  
Yifei Liu ◽  
...  
Keyword(s):  
Steady State ◽  
Membrane Proteins ◽  
Broad Spectrum ◽  
Matrix Protein ◽  
Protein Import ◽  
Nuclear Accumulation ◽  
Eukaryotic Cells ◽  
Membrane Synthesis ◽  
Peroxisomal Membrane ◽  
Peroxisome Membrane

Peroxisomes are components of virtually all eukaryotic cells. While much is known about peroxisomal matrix protein import, our understanding of how peroxisomal membrane proteins (PMPs) are targeted and inserted into the peroxisome membrane is extremely limited. Here, we show that PEX19 binds a broad spectrum of PMPs, displays saturable PMP binding, and interacts with regions of PMPs required for their targeting to peroxisomes. Furthermore, mislocalization of PEX19 to the nucleus leads to nuclear accumulation of newly synthesized PMPs. At steady state, PEX19 is bimodally distributed between the cytoplasm and peroxisome, with most of the protein in the cytoplasm. We propose that PEX19 may bind newly synthesized PMPs and facilitate their insertion into the peroxisome membrane. This hypothesis is supported by the observation that the loss of PEX19 results in degradation of PMPs and/or mislocalization of PMPs to the mitochondrion.

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.

Peroxisomal membrane proteins are properly targeted to peroxisomes in the absence of COPI- and COPII-mediated vesicular transport

2001 ◽  
Vol 114 (11) ◽  
pp. 2199-2204 ◽  
Author(s):  
Tineke Voorn-Brouwer ◽  
Astrid Kragt ◽  
Henk F. Tabak ◽  
Ben Distel
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Secretory Pathway ◽  
Human Fibroblasts ◽  
Vesicle Transport ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Classic Model ◽  
Early Secretory Pathway

The classic model for peroxisome biogenesis states that new peroxisomes arise by the fission of pre-existing ones and that peroxisomal matrix and membrane proteins are recruited directly from the cytosol. Recent studies challenge this model and suggest that some peroxisomal membrane proteins might traffic via the endoplasmic reticulum to peroxisomes. We have studied the trafficking in human fibroblasts of three peroxisomal membrane proteins, Pex2p, Pex3p and Pex16p, all of which have been suggested to transit the endoplasmic reticulum before arriving in peroxisomes. Here, we show that targeting of these peroxisomal membrane proteins is not affected by inhibitors of COPI and COPII that block vesicle transport in the early secretory pathway. Moreover, we have obtained no evidence for the presence of these peroxisomal membrane proteins in compartments other than peroxisomes and demonstrate that COPI and COPII inhibitors do not affect peroxisome morphology or integrity. Together, these data fail to provide any evidence for a role of the endoplasmic reticulum in peroxisome biogenesis.

scholarly journals PEX3 functions as a PEX19 docking factor in the import of class I peroxisomal membrane proteins

2004 ◽  
Vol 164 (6) ◽  
pp. 863-875 ◽  
Author(s):  
Yi Fang ◽  
James C. Morrell ◽  
Jacob M. Jones ◽  
Stephen J. Gould
Keyword(s):  
Membrane Proteins ◽  
Matrix Protein ◽  
Protein Import ◽  
Class Ii ◽  
Class I ◽  
Peroxisomal Membrane ◽  
Direct Role ◽  
Conserved Motif ◽  
Peroxisome Membrane ◽  
Import Receptor

PEX19 is a chaperone and import receptor for newly synthesized, class I peroxisomal membrane proteins (PMPs). PEX19 binds these PMPs in the cytoplasm and delivers them to the peroxisome for subsequent insertion into the peroxisome membrane, indicating that there may be a PEX19 docking factor in the peroxisome membrane. Here we show that PEX3 is required for PEX19 to dock at peroxisomes, interacts specifically with the docking domain of PEX19, and is required for recruitment of the PEX19 docking domain to peroxisomes. PEX3 is also sufficient to dock PEX19 at heterologous organelles and binds PEX19 via a conserved motif that is essential for this docking activity and for PEX3 function in general. Not surprisingly, transient inhibition of PEX3 abrogates class I PMP import but has no effect on class II PMP import or peroxisomal matrix protein import. Taken together, these results suggest that PEX3 plays a selective, essential, and direct role in PMP import as a docking factor for PEX19.

scholarly journals Pex13 Inactivation in the Mouse Disrupts Peroxisome Biogenesis and Leads to a Zellweger Syndrome Phenotype

2003 ◽  
Vol 23 (16) ◽  
pp. 5947-5957 ◽  
Author(s):  
Megan Maxwell ◽  
Jonas Bjorkman ◽  
Tam Nguyen ◽  
Peter Sharp ◽  
John Finnie ◽  
...  
Keyword(s):  
Protein Import ◽  
Zellweger Syndrome ◽  
Cre Recombinase ◽  
Acid Oxidation ◽  
Metabolic Dysfunction ◽  
Peroxisome Biogenesis ◽  
Peroxisomal Membrane ◽  
Severe Impairment ◽  
Biochemical Analyses ◽  
Neuronal Migration Defect

ABSTRACT Zellweger syndrome is the archetypical peroxisome biogenesis disorder and is characterized by defective import of proteins into the peroxisome, leading to peroxisomal metabolic dysfunction and widespread tissue pathology. In humans, mutations in the PEX13 gene, which encodes a peroxisomal membrane protein necessary for peroxisomal protein import, can lead to a Zellweger phenotype. To develop mouse models for this disorder, we have generated a targeted mouse with a loxP-modified Pex13 gene to enable conditional Cre recombinase-mediated inactivation of Pex13. In the studies reported here, we crossed these mice with transgenic mice that express Cre recombinase in all cells to generate progeny with ubiquitous disruption of Pex13. The mutant pups exhibited many of the clinical features of Zellweger syndrome patients, including intrauterine growth retardation, severe hypotonia, failure to feed, and neonatal death. These animals lacked morphologically intact peroxisomes and showed deficient import of matrix proteins containing either type 1 or type 2 targeting signals. Biochemical analyses of tissue and cultured skin fibroblasts from these animals indicated severe impairment of peroxisomal fatty acid oxidation and plasmalogen synthesis. The brains of these animals showed disordered lamination in the cerebral cortex, consistent with a neuronal migration defect. Thus, Pex13−/− mice reproduce many of the features of Zellweger syndrome and PEX13 deficiency in humans.

scholarly journals Defective Peroxisome Membrane Synthesis Due To Mutations in Human PEX3 Causes Zellweger Syndrome, Complementation Group G

2000 ◽  
Vol 67 (4) ◽  
pp. 967-975 ◽  
Author(s):  
Ania C. Muntau ◽  
Peter U. Mayerhofer ◽  
Barbara C. Paton ◽  
Stefan Kammerer ◽  
Adelbert A. Roscher
Keyword(s):  
Zellweger Syndrome ◽  
Complementation Group ◽  
Membrane Synthesis ◽  
Peroxisome Membrane

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.

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