Preperoxisomal vesicles can form in the absence of Pex3

We demonstrate that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells. Notably, pex3 mutant cells already contain reticular and vesicular structures that harbor key proteins of the peroxisomal receptor docking complex—Pex13 and Pex14—as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). These reticular and vesicular structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible to autophagic degradation, which is blocked in this double mutant. Contrary to earlier suggestions, peroxisomes are not formed de novo from the endoplasmic reticulum when the PEX3 gene is reintroduced in pex3 cells. Instead, we find that reintroduced Pex3 sorts to the preperoxisomal structures in pex3 cells, after which these structures mature into normal peroxisomes.

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

Molecular and Cellular Biology ◽

10.1128/mcb.18.5.2789 ◽

1998 ◽

Vol 18 (5) ◽

pp. 2789-2803 ◽

Cited By ~ 115

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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The Peroxisome Biogenesis Factors Pex4p, Pex22p, Pex1p, and Pex6p Act in the Terminal Steps of Peroxisomal Matrix Protein Import

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7516-7526.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7516-7526 ◽

Cited By ~ 117

Author(s):

Cynthia S. Collins ◽

Jennifer E. Kalish ◽

James C. Morrell ◽

J. Michael McCaffery ◽

Stephen J. Gould

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Protein Translocation ◽

Matrix Proteins ◽

Peroxisome Biogenesis ◽

Peroxisomal Membrane ◽

The Matrix ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Mutant Cells

ABSTRACT Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in mostpex mutants of the yeast Pichia pastorisbut is severely reduced in pex4 andpex22 mutants and moderately reduced in pex1and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups ofpex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1,pex6, and pex22 mutant cells, we show here thatpex4Δ mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.

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Involvement of the Endoplasmic Reticulum in Peroxisome Formation

Molecular Biology of the Cell ◽

10.1091/mbc.e02-11-0734 ◽

2003 ◽

Vol 14 (7) ◽

pp. 2900-2907 ◽

Cited By ~ 128

Author(s):

Hans J. Geuze ◽

Jean Luc Murk ◽

An K. Stroobants ◽

Janice M. Griffith ◽

Monique J. Kleijmeer ◽

...

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Dendritic Cells ◽

Three Dimensional ◽

Matrix Proteins ◽

Peroxisomal Membrane ◽

Present Evidence ◽

Transporter Protein ◽

Atp Binding Cassette Transporter ◽

The Matrix

The traditional view holds that peroxisomes are autonomous organelles multiplying by growth and division. More recently, new observations have challenged this concept. Herein, we present evidence supporting the involvement of the endoplasmic reticulum (ER) in peroxisome formation by electron microscopy, immunocytochemistry and three-dimensional image reconstruction of peroxisomes and associated compartments in mouse dendritic cells. We found the peroxisomal membrane protein Pex13p and the ATP-binding cassette transporter protein PMP70 present in specialized subdomains of the ER that were continuous with a peroxisomal reticulum from which mature peroxisomes arose. The matrix proteins catalase and thiolase were only detectable in the reticula and peroxisomes. Our results suggest the existence of a maturation pathway from the ER to peroxisomes and implicate the ER as a major source from which the peroxisomal membrane is derived.

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Peroxisomal assembly: membrane proliferation precedes the induction of the abundant matrix proteins in the methylotrophic yeast Candida boidinii

Journal of Cell Science ◽

10.1242/jcs.96.4.583 ◽

1990 ◽

Vol 96 (4) ◽

pp. 583-590

Author(s):

M. Veenhuis ◽

J.M. Goodman

Keyword(s):

Early Stage ◽

Morphological Changes ◽

Polyclonal Antibodies ◽

Alcohol Oxidase ◽

Methylotrophic Yeast ◽

Candida Boidinii ◽

Peroxisomal Membrane ◽

Cell Induction ◽

The Matrix ◽

Dihydroxyacetone Synthase

Peroxisomes are massively induced when methylotrophic yeasts are cultured in medium containing methanol. These organelles contain enzymes that catalyze the initial steps of methanol assimilation. In Candida boidinii, a methylotrophic yeast, the peroxisomal matrix (internal compartment) is composed almost exclusively of two proteins, alcohol oxidase and dihydroxyacetone synthase; catalase is present in much lower abundance. Monoclonal and polyclonal antibodies are available against peroxisomal matrix and membrane proteins. These were utilized to correlate the induction of specific proteins with the morphological changes occurring during peroxisomal proliferation. Cells cultured in glucose-containing medium contain two to five small microbodies, which are identifiable by catalase staining and immunoreactivity with a monoclonal antibody against PMP47, an integral peroxisomal membrane protein. Three stages of proliferation can be distinguished when cells are switched to methanol as the carbon source. (1) There is an early stage (within 1 h) in which several peroxisomes develop from a preexisting organelle. This is accompanied by an increase in catalase activity and an induction of PMP47, but no detectable induction of alcohol oxidase or dihydroxyacetone synthase is observed. (2) From 1 to 2.5 h there is further division of these microbodies until up to 30 small peroxisomes generally are present in each of one or two clusters per cell. Induction of alcohol oxidase, dihydroxyacetone synthase and PMP20, a protein that is distributed in the matrix and membrane, is detectable during this time. Serial sections reveal that some peroxisomes remain uninduced while others undergo proliferation. Such sections also show no obvious connections between peroxisomes within clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

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Autophagosome formation in relation to the endoplasmic reticulum

Journal of Biomedical Science ◽

10.1186/s12929-020-00691-6 ◽

2020 ◽

Vol 27 (1) ◽

Author(s):

Yo-hei Yamamoto ◽

Takeshi Noda

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

De Novo ◽

Transmembrane Protein ◽

Lipid Transfer ◽

Membrane Structures ◽

Autophagosome Formation ◽

Selective Autophagy ◽

The Relationship ◽

Er Membrane

Abstract Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.

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Capacity of the Golgi Apparatus for Biogenesis from the Endoplasmic Reticulum

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0437 ◽

2003 ◽

Vol 14 (12) ◽

pp. 5011-5018 ◽

Cited By ~ 62

Author(s):

Sapna Puri ◽

Adam D. Linstedt

Keyword(s):

Endoplasmic Reticulum ◽

Golgi Apparatus ◽

Self Assembly ◽

De Novo ◽

Brefeldin A ◽

The Other ◽

Matrix Proteins ◽

Er Export ◽

Golgi Matrix

It is unclear whether the mammalian Golgi apparatus can form de novo from the ER or whether it requires a preassembled Golgi matrix. As a test, we assayed Golgi reassembly after forced redistribution of Golgi matrix proteins into the ER. Two conditions were used. In one, ER redistribution was achieved using a combination of brefeldin A (BFA) to cause Golgi collapse and H89 to block ER export. Unlike brefeldin A alone, which leaves matrix proteins in relatively large remnant structures outside the ER, the addition of H89 to BFA-treated cells caused ER accumulation of all Golgi markers tested. In the other, clofibrate treatment induced ER redistribution of matrix and nonmatrix proteins. Significantly, Golgi reassembly after either treatment was robust, implying that the Golgi has the capacity to form de novo from the ER. Furthermore, matrix proteins reemerged from the ER with faster ER exit rates. This, together with the sensitivity of BFA remnants to ER export blockade, suggests that presence of matrix proteins in BFA remnants is due to cycling via the ER and preferential ER export rather than their stable assembly in a matrix outside the ER. In summary, the Golgi apparatus appears capable of efficient self-assembly.

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Pex17p of Saccharomyces cerevisiae Is a Novel Peroxin and Component of the Peroxisomal Protein Translocation Machinery

The Journal of Cell Biology ◽

10.1083/jcb.140.1.49 ◽

1998 ◽

Vol 140 (1) ◽

pp. 49-60 ◽

Cited By ~ 118

Author(s):

Bettina Huhse ◽

Peter Rehling ◽

Markus Albertini ◽

Lars Blank ◽

Karl Meller ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein Translocation ◽

Coiled Coil ◽

Matrix Proteins ◽

Peroxisomal Membrane ◽

Trimeric Complex ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Membrane Spanning ◽

Mutant Cells

The Saccharomyces cerevisiae pex17-1 mutant was isolated from a screen to identify mutants defective in peroxisome biogenesis. pex17-1 and pex17 null mutants fail to import matrix proteins into peroxisomes via both PTS1- and PTS2-dependent pathways. The PEX17 gene (formerly PAS9; Albertini, M., P. Rehling, R. Erdmann, W. Girzalsky, J.A.K.W. Kiel, M. Veenhuis, and W.-H Kunau. 1997. Cell. 89:83–92) encodes a polypeptide of 199 amino acids with one predicted membrane spanning region and two putative coiled-coil structures. However, localization studies demonstrate that Pex17p is a peripheral membrane protein located at the surface of peroxisomes. Particulate structures containing the peroxisomal integral membrane proteins Pex3p and Pex11p are evident in pex17 mutant cells, indicating the existence of peroxisomal remnants (“ghosts”). This finding suggests that pex17 null mutant cells are not impaired in peroxisomal membrane biogenesis. Two-hybrid studies showed that Pex17p directly binds to Pex14p, the recently proposed point of convergence for the two peroxisomal targeting signal (PTS)-dependent import pathways, and indirectly to Pex5p, the PTS1 receptor. The latter interaction requires Pex14p, indicating the potential of these three peroxins to form a trimeric complex. This conclusion is supported by immunoprecipitation experiments showing that Pex14p and Pex17p coprecipitate with both PTS receptors in the absence of Pex13p. From these and other studies we conclude that Pex17p, in addition to Pex13p and Pex14p, is the third identified component of the peroxisomal translocation machinery.

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A signal from inside the peroxisome initiates its division by promoting the remodeling of the peroxisomal membrane

The Journal of Cell Biology ◽

10.1083/jcb.200609072 ◽

2007 ◽

Vol 177 (2) ◽

pp. 289-303 ◽

Cited By ~ 41

Author(s):

Tong Guo ◽

Christopher Gregg ◽

Tatiana Boukh-Viner ◽

Pavlo Kyryakov ◽

Alexander Goldberg ◽

...

Keyword(s):

Fatty Acid ◽

Cytoskeletal Proteins ◽

Matrix Proteins ◽

Peroxisome Biogenesis ◽

Peroxisomal Membrane ◽

Membrane Fission ◽

The Matrix ◽

Complete Set ◽

Acyl Coa Oxidase ◽

Peroxisome Division

We define the dynamics of spatial and temporal reorganization of the team of proteins and lipids serving peroxisome division. The peroxisome becomes competent for division only after it acquires the complete set of matrix proteins involved in lipid metabolism. Overloading the peroxisome with matrix proteins promotes the relocation of acyl-CoA oxidase (Aox), an enzyme of fatty acid β-oxidation, from the matrix to the membrane. The binding of Aox to Pex16p, a membrane-associated peroxin required for peroxisome biogenesis, initiates the biosynthesis of phosphatidic acid and diacylglycerol (DAG) in the membrane. The formation of these two lipids and the subsequent transbilayer movement of DAG initiate the assembly of a complex between the peroxins Pex10p and Pex19p, the dynamin-like GTPase Vps1p, and several actin cytoskeletal proteins on the peroxisomal surface. This protein team promotes membrane fission, thereby executing the terminal step of peroxisome division.

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Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments

Journal of Cell Science ◽

10.1242/jcs.114.15.2863 ◽

2001 ◽

Vol 114 (15) ◽

pp. 2863-2868

Author(s):

Mary Q. Stewart ◽

Renee D. Esposito ◽

Jehangir Gowani ◽

Joel M. Goodman

Keyword(s):

Alcohol Oxidase ◽

Protein Assembly ◽

Matrix Proteins ◽

Thiamine Pyrophosphate ◽

Model Organisms ◽

Candida Boidinii ◽

Methylotrophic Yeasts ◽

The Matrix ◽

Cellular Compartments ◽

Dihydroxyacetone Synthase

Alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS) constitute the bulk of matrix proteins in methylotrophic yeasts, model organisms for the study of peroxisomal assembly. Both are homooligomers; AO is a flavin-containing octamer, whereas DHAS is a thiamine pyrophosphate-containing dimer. Experiments in recent years have demonstrated that assembly of peroxisomal oligomers can occur before import; indeed the absence of chaperones within the peroxisomal matrix calls into question the ability of this compartment to assemble proteins at all. We have taken a direct pulse-chase approach to monitor import and assembly of the two major proteins of peroxisomes in Candida boidinii. Oligomers of AO are not observed in the cytosol, consistent with the proteins inability to undergo piggyback import. Indeed, oligomerization of AO can be followed within the peroxisomal matrix, directly demonstrating the capacity of this compartment for protein assembly. By contrast, DHAS quickly dimerizes in the cytosol before import. Binding and import was slowed at 15°C; the effect on AO was more dramatic. In conclusion, our data indicate that peroxisomes assemble AO in the matrix, while DHAS undergoes dimerization prior to import.

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Cis-Golgi Matrix Proteins Move Directly to Endoplasmic Reticulum Exit Sites by Association with Tubules

Molecular Biology of the Cell ◽

10.1091/mbc.e05-05-0447 ◽

2006 ◽

Vol 17 (1) ◽

pp. 525-538 ◽

Cited By ~ 40

Author(s):

Gonzalo A. Mardones ◽

Christopher M. Snyder ◽

Kathryn E. Howell

Keyword(s):

Endoplasmic Reticulum ◽

Imaging Techniques ◽

Specific Interaction ◽

Transmembrane Proteins ◽

Matrix Proteins ◽

Tubule Formation ◽

The Matrix ◽

Er Exit Sites ◽

Retrograde Traffic ◽

Golgi Matrix

The role of cis-medial Golgi matrix proteins in retrograde traffic is poorly understood. We have used imaging techniques to understand the relationship between the cis-medial Golgi matrix and transmembrane proteins during retrograde traffic in control and brefeldin A (BFA)-treated cells. All five of the cis-medial matrix proteins tested were associated with retrograde tubules within 2-3 min of initiation of tubule formation. Then, at later time points (3-10 min), transmembrane proteins are apparent in the same tubules. Strikingly, both the matrix proteins and the transmembrane proteins moved directly to endoplasmic reticulum (ER) exit sites labeled with p58 and Sec13, and there seemed to be a specific interaction between the ER exit sites and the tips or branch points of the tubules enriched for the matrix proteins. After the initial interaction, Golgi matrix proteins accumulated rapidly (5-10 min) at ER exit sites, and Golgi transmembrane proteins accumulated at the same sites ∼2 h later. Our data suggest that Golgi cis-medial matrix proteins participate in Golgi-to-ER traffic and play a novel role in tubule formation and targeting.

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