Sorting and function of peroxisomal membrane proteins

Peroxisomes are subcellular organelles and are present in virtually all eukaryotic cells. Characteristic features of these organelles are their inducibility and their functional versatility. Their importance in the intermediary metabolism of cells is exemplified by the discovery of several inborn, fatal peroxisomal errors in man, the so-called peroxisomal disorders. Recent findings in research on peroxisome biogenesis and function have demonstrated that peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs) follow separate pathways to reach their target organelle. This paper addresses the principles of PMP sorting and summarizes the current knowledge of the role of these proteins in organelle biogenesis and function.     

Targeting signals in peroxisomal membrane proteins

Peroxisomal membrane proteins (PMPs) are encoded by the nuclear genome and translated on cytoplasmic ribosomes. Newly synthesized PMPs can be targeted directly from the cytoplasm to peroxisomes or travel to peroxisomes via the endoplasmic reticulum (ER). The mechanisms responsible for the targeting of these proteins to the peroxisomal membrane are still rather poorly understood. However, it is clear that the trafficking of PMPs to peroxisomes depends on the presence of cis-acting targeting signals, called mPTSs. These mPTSs show great variability both in the identity and number of requisite residues. An emerging view is that mPTSs consist of at least two functionally distinct domains: a targeting element, which directs the newly synthesized PMP from the cytoplasm to its target membrane, and a membrane-anchoring sequence, which is required for the permanent insertion of the protein into the peroxisomal membrane. In this review, we summarize our knowledge of the mPTSs currently identified.     

Presence of small GTP-binding proteins in the peroxisomal membrane.

Highly purified peroxisomal membranes stripped from their peripheral membrane proteins and only minimally contaminated with other membranes, contained three GTP-binding proteins of 29, 27 and 25 kDa, respectively. Bound radioactive GTP was displaced by unlabelled GTP, GTP analogs and GDP but not by GMP or other nucleotides. GTP binding was markedly decreased by trypsin treatment of intact purified peroxisomes; it increased 2-3-fold after pretreatment of the animals with a peroxisome proliferator. We conclude that the peroxisomal membrane contains small GTP-binding proteins that are exposed to the cytosol and that are firmly anchored in the membrane. We speculate that these proteins are involved in peroxisome multiplication by fission or budding during peroxisome biogenesis and proliferation.     

Yarrowia lipolytica cells mutant for the PEX24 gene encoding a peroxisomal membrane peroxin mislocalize peroxisomal proteins and accumulate membrane structures containing both peroxisomal matrix and membrane proteins

Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. Functional complementation of the oleic acid-nonutilizing strain mut1-1 of the yeast Yarrowia lipolytica has identified the novel gene, PEX24. PEX24 encodes Pex24p, a protein of 550 amino acids (61,100 Da). Pex24p is an integral membrane protein of peroxisomes that exhibits high sequence homology to two hypothetical proteins encoded by the open reading frames YHR150W and YDR479C of the Saccharomyces cerevisiae genome. Pex24p is detectable in wild-type cells grown in glucose-containing medium, and its levels are significantly increased by incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. pex24 mutants are compromised in the targeting of both matrix and membrane proteins to peroxisomes. Although pex24 mutants fail to assemble functional peroxisomes, they do harbor membrane structures that contain subsets of peroxisomal proteins.     

Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins

Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes. Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins. Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol1, 489-530). Progress has been made in the elucidation of how these proteins are targeted to the organelle. In addition, the understanding of the composition of the peroxisomal import apparatus and the order of events taking place during the cascade of peroxisomal protein import has increased significantly. However, our knowledge on the basic principles of peroxisomal membrane protein insertion or translocation of peroxisomal matrix proteins across the peroxisomal membrane is rather limited. The latter is of particular interest as the peroxisomal import machinery accommodates folded, even oligomeric, proteins, which distinguishes this apparatus from the well characterized translocons of other organelles. Furthermore, the origin of the peroxisomal membrane is still enigmatic. Recent observations suggest the existence of two classes of peroxisomal membrane proteins. Newly synthesized class I proteins are directly targeted to and inserted into the peroxisomal membrane, while class II proteins reach their final destination via the endoplasmic reticulum or a subcompartment thereof, which would be in accord with the idea that the peroxisomal membrane might be derived from the endoplasmic reticulum.     

OCTN3 is a mammalian peroxisomal membrane carnitine transporter

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20 microM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism.     

Antibodies directed against the peroxisomal targeting signal of firefly luciferase recognize multiple mammalian peroxisomal proteins

We have previously shown that the peroxisomal targeting signal in firefly luciferase consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine (Gould, S.J., G.A. Keller, N. Hosken, J. Wilkinson, and S. Subramani, 1989. J. Cell Biol. 108:1657-1664). Antibodies were raised against a synthetic peptide that contained this tripeptide at its COOH terminus. Immunofluorescence and immunocryoelectron microscopy revealed that the anti-peptide antibodies specifically detected peroxisomes in mammalian cells. Further characterization revealed that the antibodies were primarily directed against the COOH-terminal three amino acids of the peptide. In Western blot experiments, the antibodies recognized 15-20 rat liver peroxisomal proteins, but reacted with only a few proteins from other subcellular compartments. These results provide independent immunological evidence that the peroxisomal targeting signal identified in firefly luciferase is present in many peroxisomal proteins.     

Multiple distinct targeting signals in integral peroxisomal membrane proteins

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.     

Mutants of the Yarrowia lipolytica PEX23 gene encoding an integral peroxisomal membrane peroxin mislocalize matrix proteins and accumulate vesicles containing peroxisomal matrix and membrane proteins

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 yeast Saccharomyces 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.     

The novel tail-anchored membrane protein Mff controls mitochondrial and peroxisomal fission in mammalian cells

Few components of the mitochondrial fission machinery are known, even though mitochondrial fission is a complex process of vital importance for cell growth and survival. Here, we describe a novel protein that controls mitochondrial fission. This protein was identified in a small interfering RNA (siRNA) screen using Drosophila cells. The human homologue of this protein was named Mitochondrial fission factor (Mff). Mitochondria of cells transfected with Mff siRNA form a closed network similar to the mitochondrial networks formed when cells are transfected with siRNA for two established fission proteins, Drp1 and Fis1. Like Drp1 and Fis1 siRNA, Mff siRNA also inhibits fission induced by loss of mitochondrial membrane potential, it delays cytochrome c release from mitochondria and further progression of apoptosis, and it inhibits peroxisomal fission. Mff and Fis1 are both tail anchored in the mitochondrial outer membrane, but other parts of these proteins are very different and they exist in separate 200-kDa complexes, suggesting that they play different roles in the fission process. We conclude that Mff is a novel component of a conserved membrane fission pathway used for constitutive and induced fission of mitochondria and peroxisomes.     

Structure prediction of membrane proteins

There is a large gap between the number of membrane protein (MP) sequences and that of their decoded 3D structures, especially high-resolution structures, due to difficulties in crystal preparation of MPs. However, detailed knowledge of the 3D structure is required for the fundamental understanding of the function of an MP and the interactions between the protein and its inhibitors or activators. In this paper, some computational approaches that have been used to predict MP structures are discussed and compared.     

The role of membrane proteins in mammalian autophagy

Autophagy is an evolutionarily conserved degradative process that is initiated by autophagosomes, double-membrane structures that sequester cytoplasmic material and fuse with endosomes and lysosomes to become autolysosomes. Recent progress in the identification of proteins required for autophagy has led to a substantial understanding of the process involved in making an autophagosome. Mammalian Atg9, a multi-spanning transmembrane protein, is one of the possible keys to understanding how autophagosomes are formed. Current and future advances in understanding the function of mammalian Atg9 will provide a basis for further progress. In addition, the identification of so far uncharacterized transmembrane proteins which are involved in autophagy will also help to address the important questions of where, how, and why autophagosomes form.     

Guanine nucleotide-dependent carboxyl methylation of mammalian membrane proteins

A guanine nucleotide-dependent protein carboxyl methylation is demonstrated in mammalian cell membranes. The methylation of membrane proteins of Mr 20,000-23,000 requires S-adenosylmethionine, GTP or nonhydrolyzable GTP analogs, and a cytoplasmic methyltransferase. The protein methyl groups are stable at neutral pH and under basic conditions hydrolyze to produce methanol. The specific methyl acceptor proteins and methyltransferases varied between tissues and cell types, suggesting that these methylations have cell-specific functions. The guanine nucleotide-dependent carboxyl methylations provide a possible mechanism for regulating the function of GTP-binding membrane proteins in the transduction of receptor-mediated signals of eukaryotic cells.     

Topogenesis of peroxisomal proteins

Molecular and biochemical analysis of the biogenesis of peroxisomes has made rapid progress in recent years. Research on the mechanism of targeting of peroxisomal proteins has revealed that many, but not all, peroxisomal proteins have a conserved tripeptide motif in their carboxy-terminal portions which is required for entry into peroxisomes; the topogenic signal mechanism thus differs in these instances from those employed in mitochondria and endoplasmic reticulum. Other factors involved in peroxisome biogenesis are also coming to light.     

Structure determination of membrane proteins by NMR spectroscopy.

Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splittings observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H-15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples. One-dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples. Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination.     

Distinct requirements for intra-ER sorting and budding of peroxisomal membrane proteins from the ER

During de novo peroxisome biogenesis, importomer complex proteins sort via two preperoxisomal vesicles (ppVs). However, the sorting mechanisms segregating peroxisomal membrane proteins to the preperoxisomal endoplasmic reticulum (pER) and into ppVs are unknown. We report novel roles for Pex3 and Pex19 in intra–endoplasmic reticulum (ER) sorting and budding of the RING-domain peroxins (Pex2, Pex10, and Pex12). Pex19 bridged the interaction at the ER between Pex3 and RING-domain proteins, resulting in a ternary complex that was critical for the intra-ER sorting and subsequent budding of the RING-domain peroxins. Although the docking subcomplex proteins (Pex13, Pex14, and Pex17) also required Pex19 for budding from the ER, they sorted to the pER independently of Pex3 and Pex19 and were spatially segregated from the RING-domain proteins. We also discovered a unique role for Pex3 in sorting Pex10 and Pex12, but with the docking subcomplex. Our study describes an intra-ER sorting process that regulates segregation, packaging, and budding of peroxisomal importomer subcomplexes, thereby preventing their premature assembly at the ER.     

Synthesis and subcellular location of peroxisomal membrane proteins in a peroxisome-deficient mutant of the yeast Hansenula polymorpha.

We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous culture on a glucose/methanol mixture, indicated that various PMPs were normally synthesized. As in wild type (WT) cells, the levels of PMP synthesis appeared to be dependent on specific cultivation conditions, e.g. the carbon source used for growth. In contrast to WT controls, PMPs in methanol-induced per mutants were not subject to proteolytic degradation. Biochemical and immuno(cyto)chemical studies suggested that the PMPs in methanol-induced per cells were located in small proteinaceous aggregates, separated from peroxisomal matrix proteins that were also present in the cytosol. Vesicular membranous structures, resembling the morphology of intact peroxisomes, were never detected irrespective of the growth conditions employed.     

Interaction of calmodulin and other calcium-modulated proteins with mammalian and arthropod junctional membrane proteins

Calmodulin and other calcium-modulated proteins bind in vitro to purified junctional polypeptides from rat liver gap junctions, bovine lens fiber junctions, a chymotryptic fragment from bovine lens junctions, and crayfish hepatopancreas gap junctions. The potential biological relevance of the interaction of calmodulin with junctional proteins is suggested by immunocytochemical localization of endogenous calmodulin in cortical regions of the cell where gap junctions exist. These observations provide a molecular basis for understanding the potential regulatory role of calmodulin on cell-cell communication channels in vivo. In addition, the calmodulin binding represents the first molecular homology that has been found for junctional channel proteins from mammalian and arthropod tissues.