scholarly journals An oligomeric protein is imported into peroxisomes in vivo.

1994 ◽  
Vol 127 (5) ◽  
pp. 1245-1257 ◽  
Author(s):  
J A McNew ◽  
J M Goodman
Keyword(s):  
Matrix Proteins ◽  
Animal Cells ◽  
Peroxisomal Membrane ◽  
Epitope Tag ◽  
Membrane Pores ◽  
Mild Conditions ◽  
Oligomeric Protein ◽  
The Matrix ◽  
Peroxisomal Targeting

The mechanism of translocation of peroxisomal proteins from the cytoplasm into the matrix is largely unknown. We have been studying this problem in yeast. We show that the peroxisomal targeting sequences SKL or AKL, with or without a spacer of nine glycines (G9), are sufficient to target chloramphenicol acetyltransferase (CAT) to peroxisomes of Saccharomyces cerevisiae in vivo. The mature form of CAT is a homotrimer, and complete trimerization of CAT was found to occur within a few minutes of synthesis. In contrast, import, measured by immunoelectron microscopy and organellar fractionation, occurred over several hours. To confirm that import of preassembled CAT trimers was occurring, we co-expressed CAT-G9-AKL with CAT lacking a peroxisomal targeting sequence but containing a hemagglutinin-derived epitope tag (HA-CAT). We found that HA-CAT was not imported unless it was co-expressed with CAT-G9-AKL. Both proteins were released from the organelles under mild conditions (pH 8.5) that released other matrix proteins, indicating that import had occurred. These results strongly suggested that HA-CAT was imported as a heterotrimer with CAT-G9-AKL. The process of oligomeric import also occurs in animal cells. When HA-CAT was co-expressed with CAT-G9-AKL in CV-1 cells, HA-CAT co-localized with peroxisomes but was cytoplasmic when expressed alone. It is not clear whether the import of globular proteins into peroxisomes occurs through peroxisomal membrane pores or involves membrane internalization. Both possibilities are discussed.

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

2000 ◽  
Vol 20 (20) ◽  
pp. 7516-7526 ◽  
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.

scholarly journals Enlarged Peroxisomes Are Present in Oleic Acid–grown Yarrowia lipolytica Overexpressing the PEX16 Gene Encoding an Intraperoxisomal Peripheral Membrane Peroxin

1997 ◽  
Vol 137 (6) ◽  
pp. 1265-1278 ◽  
Author(s):  
Gary A. Eitzen ◽  
Rachel K. Szilard ◽  
Richard A. Rachubinski
Keyword(s):  
Oleic Acid ◽  
Yarrowia Lipolytica ◽  
Consensus Sequence ◽  
Wild Type ◽  
Gene Encoding ◽  
Peroxisomal Membrane ◽  
Peripheral Protein ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signal

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.

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

1998 ◽  
Vol 140 (1) ◽  
pp. 49-60 ◽  
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.

scholarly journals The unique degradation pathway of the PTS2 receptor, Pex7, is dependent on the PTS receptor/coreceptor, Pex5 and Pex20

2014 ◽  
Vol 25 (17) ◽  
pp. 2634-2643 ◽  
Author(s):  
Danielle Hagstrom ◽  
Changle Ma ◽  
Soumi Guha-Polley ◽  
Suresh Subramani
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Degradation Pathway ◽  
Matrix Proteins ◽  
Receptor Recycling ◽  
Wild Type ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signals ◽  
The Stability

Peroxisomal matrix protein import uses two peroxisomal targeting signals (PTSs). Most matrix proteins use the PTS1 pathway and its cargo receptor, Pex5. The PTS2 pathway is dependent on another receptor, Pex7, and its coreceptor, Pex20. We found that during the matrix protein import cycle, the stability and dynamics of Pex7 differ from those of Pex5 and Pex20. In Pichia pastoris, unlike Pex5 and Pex20, Pex7 is constitutively degraded in wild-type cells but is stabilized in pex mutants affecting matrix protein import. Degradation of Pex7 is more prevalent in cells grown in methanol, in which the PTS2 pathway is nonessential, in comparison with oleate, suggesting regulation of Pex7 turnover. Pex7 must shuttle into and out of peroxisomes before it is polyubiquitinated and degraded by the proteasome. The shuttling of Pex7, and consequently its degradation, is dependent on the receptor recycling pathways of Pex5 and Pex20 and relies on an interaction between Pex7 and Pex20. We also found that blocking the export of Pex20 from peroxisomes inhibits PTS1-mediated import, suggesting sharing of limited components in the export of PTS receptors/coreceptors. The shuttling and stability of Pex7 are divergent from those of Pex5 and Pex20, exemplifying a novel interdependence of the PTS1 and PTS2 pathways.

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

2007 ◽  
Vol 177 (2) ◽  
pp. 289-303 ◽  
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.

scholarly journals Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor.

1996 ◽  
Vol 135 (1) ◽  
pp. 85-95 ◽  
Author(s):  
S J Gould ◽  
J E Kalish ◽  
J C Morrell ◽  
J Bjorkman ◽  
A J Urquhart ◽  
...  
Keyword(s):  
Steady State ◽  
Matrix Proteins ◽  
Cytoplasmic Protein ◽  
Peroxisomal Membrane ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisome Membrane ◽  
Peroxisomal Targeting Signal

Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (< 5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by approximately 40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pex13p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.

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 Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase.

1988 ◽  
Vol 107 (6) ◽  
pp. 2679-2688 ◽  
Author(s):  
W L Dentler
Keyword(s):  
Membrane Proteins ◽  
Atpase Activity ◽  
Tetrahymena Thermophila ◽  
High Molecular Mass ◽  
Integral Membrane Proteins ◽  
Matrix Proteins ◽  
Membrane Atpase ◽  
The Matrix ◽  
Triton X

Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.

Proteins From Oyster Shell: Biomineralization Regulators and Commercial Polymer Analogs

1999 ◽  
Vol 599 ◽  
Author(s):  
A. P. Wheeler ◽  
C. S. Sikes
Keyword(s):  
Crystal Growth ◽  
Aspartic Acid ◽  
Large Scale ◽  
Matrix Protein ◽  
Gene Families ◽  
Eastern Oyster ◽  
Matrix Proteins ◽  
The Matrix

AbstractMolluscan shell is a composite made up of μm-sized CaCO3 crystals and an organic phase (matrix). This report outlines our studies on the structure and activities of matrix proteins isolated from the inner calcite layer of shell of the Eastern oyster, including their cellular origin and structure and their relationship to the crystalline mineral phase. In addition, we present results of the synthesis and commercialization of polypeptide polymers which are based on the structure and activities of the oyster proteins. Extracted shell proteins are polyanionic and range in size from relatively small soluble forms to those which are crosslinked and insoluble. The soluble forms are capable of adsorbing to calcite in vitro and in the process changing its growth habit and acting as threshold growth inhibitors. Their function in vivo is not understood, but they may serve to control shell crystal morphology. The insoluble protein forms gels readily and may serve to provide resiliency to the shell and, from in vitro and in situ observations, appears to serve as a site for nucleation of crystals. However, from studies in vitro, these gels do not lower the energy of activation for nucleation, as previously expected. Matrix protein aggregates are identifiable by AFM on the surface of crystals, but as such do not serve as nucleation sites for new crystal growth. If the aggregates are removed, then ectopic crystal growth proceeds readily revealing orientation of the underlying crystals. All the matrix proteins contain domains rich in aspartic acid, are heavily phosphorylated, crossreact in antibody studies and may belong to a limited number of gene families with individuals modified post-synthesis. The proteins are made by a specialized group of cells located primarily some distance from the growing edge of the shell and appear to be assembled into sheets soon after secretion. Soluble anti-scalants and crosslinked insoluble water absorbents have been developed based on the structure and activity of the matrix proteins. These are primarily poly(aspartates) which can be made in large scale via thermal polycondensation of aspartic acid. The soluble forms are commercially used as biodegradable water treatment chemicals among other applications.

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