Protein Translocation into Peroxisomes

Abstract

INTRODUCTIONPeroxisomes, as well as the signals and the machinery that target proteins to this organelle, are conserved in most eukaryotic organisms. Different peroxisomal targeting signals (PTSs), 1The abbreviations used are: PTSperoxisomal targeting signalERendoplasmic reticulummPTSmembrane peroxisomal targeting signalPMPperoxisomal membrane proteinTMDtransmembrane domain. acting in concert with specific PTS receptors, account for the specific accumulation of proteins in the peroxisomal matrix and membrane. This review focuses only on the signals, proteins, and mechanisms involved in peroxisomal protein import and peroxisome biogenesis, emphasizing features unique to this organelle. The relevance of these import mechanisms to human peroxisomal disorders is reviewed elsewhere (1Rachubinski R.A. Subramani S. How proteins penetrate peroxisomes, Cell. 1995; 83: 525-528Google Scholar, 2Subramani S. Curr. Opin. Cell Biol. 1996; 8: 513-518Crossref PubMed Scopus (41) Google Scholar). The gene and protein names used in this article conform to the new nomenclature system adopted recently (3Distel B. Erdmann R. Gould S.J. Blobel G. Crane D.I. Cregg J.M. Dodt G. Fujiki Y. Goodman J.M. Just W.W. Kiel J.A.K.W. Kunau W.-H. Lazarow P.B. Mannaerts G.P. Moser H. Osumi T. Rachubinski R.A. Roscher A. Subramani S. Tabak H.F. Valle D. van der Klei I. van Veldhoven P.P. Veenhuis M. J. Cell Biol. 1996; 135: 1-3Crossref PubMed Scopus (313) Google Scholar).The absence of DNA in peroxisomes requires that all peroxisomal proteins be encoded by the nuclear genome. There is very strong evidence that many, if not all, peroxisomal matrix proteins are synthesized on free cytosolic polysomes and then imported post-translationally from the cytosol directly to the peroxisomes (1Rachubinski R.A. Subramani S. How proteins penetrate peroxisomes, Cell. 1995; 83: 525-528Google Scholar, 2Subramani S. Curr. Opin. Cell Biol. 1996; 8: 513-518Crossref PubMed Scopus (41) Google Scholar). Matrix proteins devoid of a PTS, or containing a non-functional PTS, are generally cytosolic, with the exception that certain matrix protein subunits lacking a PTS can enter peroxisomes by association with other subunits possessing a functional PTS (4Glover J.R. Andrews D.W. Rachubinski R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10541-10545Crossref PubMed Scopus (244) Google Scholar, 5McNew J.A. Goodman J.M. J. Cell Biol. 1994; 127: 1245-1257Crossref PubMed Scopus (278) Google Scholar).Most, but not all, peroxisomal membrane proteins (PMPs) are believed to be synthesized on free polysomes and then transported from the cytosol directly to the peroxisomes. In vivo evidence for direct import from the cytosol to peroxisomes exists, to my knowledge, only for one protein, PMP70 (6Imanaka T. Shiina Y. Takano T. Hashimoto T. Osumi T. J. Biol. Chem. 1996; 271: 3706-3713Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). A few PMPs have been shown to be translated on free polysomes (7Fujiki Y. Rachubinski R.A. Lazarow P.B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7127-7131Crossref PubMed Scopus (146) Google Scholar, 8Suzuki Y. Orii T. Takiguchi M. Mori M. Hijikata M. Hashimoto T. J. Biochem. (Tokyo). 1987; 101: 491-496Crossref PubMed Scopus (49) Google Scholar, 9Bodnar A.G. Rachubinski R.A. Biochem. Cell Biol. 1991; 69: 499-508Crossref PubMed Scopus (49) Google Scholar), but direct transfer from the cytosol to peroxisomes is either inferred or supported by in vitro experiments for only two proteins, PMP22 and PMP70 (6Imanaka T. Shiina Y. Takano T. Hashimoto T. Osumi T. J. Biol. Chem. 1996; 271: 3706-3713Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 10Diestelkotter P. Just W.W. J. Cell Biol. 1993; 123: 1717-1725Crossref PubMed Scopus (62) Google Scholar). The implications of these studies are discussed later.Pathways for Import of Matrix and Membrane Proteins into PeroxisomesMultiple PTSs (generally one/protein) direct proteins to the peroxisomal matrix. The most widely used of these is the conserved, C-terminal tripeptide (SKL and its functional variants) named PTS1 (1Rachubinski R.A. Subramani S. How proteins penetrate peroxisomes, Cell. 1995; 83: 525-528Google Scholar, 2Subramani S. Curr. Opin. Cell Biol. 1996; 8: 513-518Crossref PubMed Scopus (41) Google Scholar). This sequence requires the -COOH group of the last amino acid and consequently functions only at the C termini of proteins and not at internal locations. Its location explains the post-translational nature of peroxisomal protein import for PTS1-dependent polypeptides. The PTS1 sequence is recognized specifically and with high affinity (Kd = 460-500 nM) by a receptor encoded by the PpPEX5 (former name, PpPAS8) gene of the yeast Pichia pastoris (11McCollum D. Monosov E. Subramani S. J. Cell Biol. 1993; 121: 761-774Crossref PubMed Scopus (207) Google Scholar) and the HsPEX5 (former names, PTS1R and PXR1) gene in humans (12Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (383) Google Scholar, 13Fransen M. Brees C. Baumgart E. Vanhooren J.C. Baes M. Mannaerts G.P. Van Veldhoven P. J. Biol. Chem. 1995; 270: 7731-7736Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 14Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar). Homologues of these genes have been also described in Saccharomyces cerevisiae (15Van der Leij I. Franse M.M. Elgersma Y. Distel B. Tabak H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11782-11786Crossref PubMed Scopus (202) Google Scholar), Hansenula polymorpha (16Van der Klei I.J. Hilbrands R.E. Swaving G.J. Waterham H.R. Vrieling E.G. Titorenko V.I. Cregg J.M. Harder W. Veenhuis M. J. Biol. Chem. 1995; 270: 17229-17236Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and Yarrowia lipolytica (17Szilard R.K. Titorenko V.I. Veenhuis M. Rachubinski R.A. J. Cell Biol. 1995; 131: 1453-1469Crossref PubMed Scopus (97) Google Scholar).A second PTS, used by a smaller subset of peroxisomal matrix proteins, is the conserved N-terminal nonapeptide ((R/K)(L/V/I)X5(H/Q)(L/A)) (1Rachubinski R.A. Subramani S. How proteins penetrate peroxisomes, Cell. 1995; 83: 525-528Google Scholar, 2Subramani S. Curr. Opin. Cell Biol. 1996; 8: 513-518Crossref PubMed Scopus (41) Google Scholar). In contrast to the PTS1 sequence, this sequence also functions as a PTS at internal locations in proteins. 2K. N. Faber and S. Subramani, unpublished data. The PTS2 motif in proteins is recognized by its cognate receptor encoded by the ScPEX7 (former names, ScPAS7 and ScPEB1) gene of S. cerevisiae (18Marzioch M. Erdmann R. Veenhuis M. Kunau W.H. EMBO J. 1994; 13: 4908-4918Crossref PubMed Scopus (256) Google Scholar, 19Zhang J.W. Lazarow P.B. J. Cell Biol. 1995; 129: 65-80Crossref PubMed Scopus (121) Google Scholar, 20Zhang J.W. Lazarow P.B. J. Cell Biol. 1996; 132: 325-334Crossref PubMed Scopus (80) Google Scholar, 21Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar).Other sequences from peroxisomal matrix proteins, fulfilling the criterion of being sufficient for peroxisomal targeting of a reporter protein, have been described (cited in Ref. 2Subramani S. Curr. Opin. Cell Biol. 1996; 8: 513-518Crossref PubMed Scopus (41) Google Scholar). With one exception (22Elgersma Y. van Roermund C.W. Wanders R.J. Tabak H.F. EMBO J. 1995; 14: 3472-3479Crossref PubMed Scopus (161) Google Scholar), it is not yet clear, however, that these are true PTSs in the sense that there are distinct receptors for them. They are poorly defined, and their use by other peroxisomal proteins is unconfirmed.Sequences responsible for the sorting of proteins to the peroxisomal membrane have been identified only recently. These membrane PTSs (mPTSs) have been defined in only two proteins. Goodman and colleagues (23McNew J.A. Goodman J.M. Trends Biochem. Sci. 1996; 21: 54-58Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 24Dyer J.M. McNew J.A. Goodman J.M. J. Cell Biol. 1996; 133: 269-280Crossref PubMed Scopus (145) Google Scholar) have narrowed an mPTS to a 20-amino acid loop facing the peroxisomal matrix in the peroxisomal membrane protein, PMP47, from Candida boidinii. This loop is located between putative transmembrane domains (TMDs) in a protein postulated to have six TMDs (23McNew J.A. Goodman J.M. Trends Biochem. Sci. 1996; 21: 54-58Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 24Dyer J.M. McNew J.A. Goodman J.M. J. Cell Biol. 1996; 133: 269-280Crossref PubMed Scopus (145) Google Scholar). Another mPTS has been defined in the first 40 amino acids of P. pastoris PpPex3p (former name, PpPas2p) and also does not include any TMDs (25Wiemer E.A.C. Lüers G. Faber K.N. Wenzel T. Veenhuis M. Subramani S. J. Biol. Chem. 1996; 271: 18973-18980Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Receptors for these mPTSs are not known.Mutants affecting protein import via the PTS1 (11McCollum D. Monosov E. Subramani S. J. Cell Biol. 1993; 121: 761-774Crossref PubMed Scopus (207) Google Scholar, 12Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (383) Google Scholar, 14Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar, 15Van der Leij I. Franse M.M. Elgersma Y. Distel B. Tabak H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11782-11786Crossref PubMed Scopus (202) Google Scholar, 16Van der Klei I.J. Hilbrands R.E. Swaving G.J. Waterham H.R. Vrieling E.G. Titorenko V.I. Cregg J.M. Harder W. Veenhuis M. J. Biol. Chem. 1995; 270: 17229-17236Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 17Szilard R.K. Titorenko V.I. Veenhuis M. Rachubinski R.A. J. Cell Biol. 1995; 131: 1453-1469Crossref PubMed Scopus (97) Google Scholar) or PTS2 (18Marzioch M. Erdmann R. Veenhuis M. Kunau W.H. EMBO J. 1994; 13: 4908-4918Crossref PubMed Scopus (256) Google Scholar, 19Zhang J.W. Lazarow P.B. J. Cell Biol. 1995; 129: 65-80Crossref PubMed Scopus (121) Google Scholar) pathways alone as well as mutants deficient in protein import via both of these pathways (reviewed in Ref. 23McNew J.A. Goodman J.M. Trends Biochem. Sci. 1996; 21: 54-58Abstract Full Text PDF PubMed Scopus (147) Google Scholar) are known. In many of these mutants peroxisomal remnants or membrane “ghosts” are seen, suggesting that membrane proteins are sorted via a different route. Mutants deficient in the import of PMPs are expected to have no peroxisomal ghosts and should be compromised in both peroxisome biogenesis and matrix protein import (25Wiemer E.A.C. Lüers G. Faber K.N. Wenzel T. Veenhuis M. Subramani S. J. Biol. Chem. 1996; 271: 18973-18980Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar).Involvement of the PTS Receptors in Peroxisomal Protein ImportThere is genetic and biochemical evidence that the PTS receptors are essential for import of proteins to the matrix, but not the membrane, of peroxisomes. Mutations in the PTS1 receptor (PEX5) gene (or its homologues) in yeasts and humans exhibit a specific impairment in the import of PTS1- but not of PTS2-containing proteins (11McCollum D. Monosov E. Subramani S. J. Cell Biol. 1993; 121: 761-774Crossref PubMed Scopus (207) Google Scholar, 12Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (383) Google Scholar, 14Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar, 15Van der Leij I. Franse M.M. Elgersma Y. Distel B. Tabak H.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11782-11786Crossref PubMed Scopus (202) Google Scholar, 16Van der Klei I.J. Hilbrands R.E. Swaving G.J. Waterham H.R. Vrieling E.G. Titorenko V.I. Cregg J.M. Harder W. Veenhuis M. J. Biol. Chem. 1995; 270: 17229-17236Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 17Szilard R.K. Titorenko V.I. Veenhuis M. Rachubinski R.A. J. Cell Biol. 1995; 131: 1453-1469Crossref PubMed Scopus (97) Google Scholar). Conversely, mutations in the PTS2 receptor (PEX7) gene compromise only the import of PTS2-containing proteins (18Marzioch M. Erdmann R. Veenhuis M. Kunau W.H. EMBO J. 1994; 13: 4908-4918Crossref PubMed Scopus (256) Google Scholar, 19Zhang J.W. Lazarow P.B. J. Cell Biol. 1995; 129: 65-80Crossref PubMed Scopus (121) Google Scholar).In biochemical experiments, the yeast and human PTS1 receptors bind the PTS1 peptide specifically (14Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar, 26Terlecky S.R. Nuttley W.M. McCollum D. Sock E. Subramani S. EMBO J. 1995; 14: 3627-3634Crossref PubMed Scopus (155) Google Scholar, 27Wiemer E.A.C. Terlecky S.R. Nuttley W.M. Subramani S. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 637-648Crossref PubMed Scopus (6) Google Scholar). Antibodies to HsPex5p (human PTS1R) inhibit the peroxisomal import of a PTS1-containing reporter protein in permeabilized cells (14Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar). Interactions have been detected in a yeast two-hybrid system between yeast and human PTS1 receptors, on the one hand, and PTS1-containing proteins on the other (13Fransen M. Brees C. Baumgart E. Vanhooren J.C. Baes M. Mannaerts G.P. Van Veldhoven P. J. Biol. Chem. 1995; 270: 7731-7736Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 22Elgersma Y. van Roermund C.W. Wanders R.J. Tabak H.F. EMBO J. 1995; 14: 3472-3479Crossref PubMed Scopus (161) Google Scholar). Similar interactions have been detected between the PTS2 receptor and PTS2-containing proteins (20Zhang J.W. Lazarow P.B. J. Cell Biol. 1996; 132: 325-334Crossref PubMed Scopus (80) Google Scholar, 21Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar). Evidence for this ranges from genetic suppression of a temperature-sensitive mutation in a yeast PTS2 sequence by overexpression of the receptor ScPex7p (previously ScPas7p) to two-hybrid interactions between the receptor and PTS2-containing proteins and coimmunoprecipitation of the receptor-ligand complex (20Zhang J.W. Lazarow P.B. J. Cell Biol. 1996; 132: 325-334Crossref PubMed Scopus (80) Google Scholar, 21Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar). Proof of direct binding between the PTS2 receptor and its ligand is not yet available.Consistent with the ability of the PTS2 sequence to function as a targeting signal in vivo when placed at internal locations in proteins,2 the PTS2 receptor (ScPex7p) of S. cerevisiae binds to a Gal4p-thiolase fusion in which the PTS2 is located internally (21Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar).An earlier study (14Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar) had noted that a human patient with a nonsense mutation in the PTS1R gene was deficient in the import of both PTS1- and PTS2-containing proteins, suggesting a connection between the PTS1 and PTS2 import pathways. Because proteins containing tetratricopeptide repeats are known to interact with polypeptides possessing WD40 repeats, this connection had been explained by a possible association between the HsPex5p (a tetratricopeptide repeat protein) and the human PTS2 receptor (HsPex7p, a putative WD40 protein based on homology with ScPex7p) (1Rachubinski R.A. Subramani S. How proteins penetrate peroxisomes, Cell. 1995; 83: 525-528Google Scholar, 27Wiemer E.A.C. Terlecky S.R. Nuttley W.M. Subramani S. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 637-648Crossref PubMed Scopus (6) Google Scholar). Proof of such an association between the PTS1 (ScPex5p) and PTS2 receptors (ScPex7p) of S. cerevisiae was obtained recently in a yeast two-hybrid system (21Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar).Controversial Subcellular Locations of the PTS1 and PTS2 ReceptorsIn cell fractionation studies, the PTS1 receptor of P. pastoris, PpPex5p, behaves like an integral PMP though no TMDs are obvious in its sequence. It faces the cytosol and binds the PTS1 sequence directly with high affinity, both as a purified recombinant protein and when it is associated with purified peroxisomes (26Terlecky S.R. Nuttley W.M. McCollum D. Sock E. Subramani S. EMBO J. 1995; 14: 3627-3634Crossref PubMed Scopus (155) Google Scholar). However, multiple subcellular locations have been found for its homologues from other species. The S. cerevisiae homologue, ScPex5p, is mainly cytosolic (28Elgersma Y. Kwast L. Klein A. Voorn-Brouwer T. Van den Berg M. Metzig B. America T. Tabak H.F. Distel B. J. Cell Biol. 1996; 135: 97-109Crossref PubMed Scopus (183) Google Scholar), the HpPex5p (former names, HpPah2p and HpPer3p) from H. polymorpha is cytosolic and intraperoxisomal (16Van der Klei I.J. Hilbrands R.E. Swaving G.J. Waterham H.R. Vrieling E.G. Titorenko V.I. Cregg J.M. Harder W. Veenhuis M. J. Biol. Chem. 1995; 270: 17229-17236Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 29Nuttley W.M. Szilard R.K. Smith J.J. Veenhuis M. Rachubinski R.A. Gene (Amst.). 1995; 160: 33-39Crossref PubMed Scopus (29) Google Scholar), and YlPex5p (former name, YlPay32p) from Y. lipolytica is principally intraperoxisomal (17Szilard R.K. Titorenko V.I. Veenhuis M. Rachubinski R.A. J. Cell Biol. 1995; 131: 1453-1469Crossref PubMed Scopus (97) Google Scholar). HsPex5p (former names, PTS1R and PXR1) has been described as being mainly cytosolic and only partially peroxisomal (12Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (383) Google Scholar, 14Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar), as well as completely peroxisome-associated (13Fransen M. Brees C. Baumgart E. Vanhooren J.C. Baes M. Mannaerts G.P. Van Veldhoven P. J. Biol. Chem. 1995; 270: 7731-7736Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar).The location of the PTS2 receptor in S. cerevisiae is equally equivocal. Kunau and colleagues (18Marzioch M. Erdmann R. Veenhuis M. Kunau W.H. EMBO J. 1994; 13: 4908-4918Crossref PubMed Scopus (256) Google Scholar, 21Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar) report that an epitope-tagged and overexpressed version of ScPex7p is mainly cytosolic and partially peroxisomal, whereas Zhang and Lazarow (19Zhang J.W. Lazarow P.B. J. Cell Biol. 1995; 129: 65-80Crossref PubMed Scopus (121) Google Scholar, 20Zhang J.W. Lazarow P.B. J. Cell Biol. 1996; 132: 325-334Crossref PubMed Scopus (80) Google Scholar) find that a different epitope-tagged version of the same protein is intraperoxisomal and that the protein has a PTS near its N terminus.Shuttling of Receptors between the Cytosol and the PeroxisomesThe varying subcellular locations of the PTS1 and PTS2 receptors have resulted in a model that proposes shuttling of the receptors between the cytosol and the peroxisomes (1Rachubinski R.A. Subramani S. How proteins penetrate peroxisomes, Cell. 1995; 83: 525-528Google Scholar). It is clear that both the PTS1 and PTS2 receptors are capable of binding their respective PTSs in the absence of peroxisomes (11McCollum D. Monosov E. Subramani S. J. Cell Biol. 1993; 121: 761-774Crossref PubMed Scopus (207) Google Scholar, 12Dodt G. Braverman N. Wong C. Moser A. Moser H.W. Watkins P. Valle D. Gould S.J. Nat. Genet. 1995; 9: 115-125Crossref PubMed Scopus (383) Google Scholar, 13Fransen M. Brees C. Baumgart E. Vanhooren J.C. Baes M. Mannaerts G.P. Van Veldhoven P. J. Biol. Chem. 1995; 270: 7731-7736Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 14Wiemer E.A. Nuttley W.M. Bertolaet B.L. Li X. Francke U. Wheelock M.J. Anne U.K. Johnson K.R. Subramani S. J. Cell Biol. 1995; 130: 51-65Crossref PubMed Scopus (164) Google Scholar, 21Rehling P. Marzioch M. Niesen F. Wittke E. Veenhuis M. Kunau W. EMBO J. 1996; 15: 2901-2913Crossref PubMed Scopus (141) Google Scholar, 26Terlecky S.R. Nuttley W.M. McCollum D. Sock E. Subramani S. EMBO J. 1995; 14: 3627-3634Crossref PubMed Scopus (155) Google Scholar, 27Wiemer E.A.C. Terlecky S.R. Nuttley W.M. Subramani S. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 637-648Crossref PubMed Scopus (6) Google Scholar). Thus, cytosolic PTS receptors would be capable of binding PTS-containing proteins. Following this initial interaction, the receptor(s) would transport the PTS-containing protein for delivery to the translocation machinery in the plane of the peroxisomal membrane (18Marzioch M. Erdmann R. Veenhuis M. Kunau W.H. EMBO J. 1994; 13: 4908-4918Crossref PubMed Scopus (256) Google Scholar). The sequence of events after this step remains a mystery. Several scenarios can be entertained. 1) The receptor might be recycled to the cytosol for another round of import. 2) The receptor might enter the peroxisome with its cargo and then be degraded within the matrix following release of the cargo. 3) The receptor might deliver its cargo in the peroxisomal matrix and then be recycled out of the peroxisome. Experimental tests need to be designed to address which of these possibilities is correct.In Vitro Systems for the Analysis of ImportThe import of radiolabeled matrix proteins translated in vitro into rat liver and yeast peroxisomes has been monitored using binding and protease protection assays (30Imanaka T. Small G.M. Lazarow P.B. J. Cell Biol. 1987; 105: 2915-2922Crossref PubMed Scopus (150) Google Scholar, 31Miyazawa S. Osumi T. Hashimoto T. Ohno K. Miura S. Fujiki Y. Mol. Cell. Biol. 1989; 9: 83-91Crossref PubMed Scopus (186) Google Scholar, 32Thieringer R. Shio H. Han Y.S. Cohen G. Lazarow P.B. Mol. Cell. Biol. 1991; 11: 510-522Crossref PubMed Scopus (62) Google Scholar, 33Miura S. Miyazawa S. Osumi T. Hashimoto T. Fujiki Y. J. Biochem. (Tokyo). 1994; 115: 1064-1068Crossref PubMed Scopus (23) Google Scholar). The import was time-, temperature-, ATP-, and signal-dependent.Import of PTS1-containing proteins into permeabilized mammalian cells has shown, in addition to the features described above, that the process is cytosol-dependent and sensitive to N-ethylmaleimide (34Wendland M. Subramani S. J. Cell Biol. 1993; 120: 675-685Crossref PubMed Scopus (103) Google Scholar, 35Rapp S. Soto U. Just W.W. Exp. Cell Res. 1993; 205: 59-65Crossref PubMed Scopus (39) Google Scholar).One report of in vitro import of a PTS2-containing protein into a subcellular fraction enriched in peroxisomes has appeared (33Miura S. Miyazawa S. Osumi T. Hashimoto T. Fujiki Y. J. Biochem. (Tokyo). 1994; 115: 1064-1068Crossref PubMed Scopus (23) Google Scholar), but the specific requirements have not been elucidated.Quantitative cytosol-dependent in vitro import assays are not yet available. The fluorescence-based assays in permeabilized cells are not quantitative, and the import assays using proteins radiolabeled by in vitro translation are cytosol-independent.PMPs can also be imported post-translationally into purified rat liver peroxisomes in vitro (6Imanaka T. Shiina Y. Takano T. Hashimoto T. Osumi T. J. Biol. Chem. 1996; 271: 3706-3713Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 10Diestelkotter P. Just W.W. J. Cell Biol. 1993; 123: 1717-1725Crossref PubMed Scopus (62) Google Scholar). Insertion in the membrane was evaluated using extractability with alkaline sodium carbonate. The insertion of PMP22 and PMP70 into rat liver peroxisomes is time- and temperature-dependent but independent of ATP and N-ethylmaleimide-sensitive factors (6Imanaka T. Shiina Y. Takano T. Hashimoto T. Osumi T. J. Biol. Chem. 1996; 271: 3706-3713Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 10Diestelkotter P. Just W.W. J. Cell Biol. 1993; 123: 1717-1725Crossref PubMed Scopus (62) Google Scholar). It is not yet clear, however, whether the topology of the proteins inserted in the membrane mimics that observed in vivo. The mPTSs are unknown for the proteins studied, so the signal dependence of import could not be assessed.Cytosolic and Membrane Proteins Involved in ImportCytosolic hsc70 (the homologue of Escherichia coli DnaK) and hsc40 (homologue of E. coli DnaJ) are required for import of PTS1-containing proteins in permeabilized cells (36Walton P.A. Wendland M. Subramani S. Rachubinski R.A. Welch W.J. J. Cell Biol. 1994; 125: 1037-1046Crossref PubMed Scopus (111) Google Scholar). 3W. Nuttley and S. Subramani, unpublished data. Because protein unfolding (see later) is not essential for import of peroxisomal matrix proteins, the requirement for these factors has been explained in terms of alternative models (2Subramani S. Curr. Opin. Cell Biol. 1996; 8: 513-518Crossref PubMed Scopus (41) Google Scholar), which do not require the stabilization of fully unfolded proteins by these chaperones.It is likely that in mammalian cells, and in several yeasts, the PTS1 and PTS2 receptors shuttle between the cytosol and the peroxisomal membrane, transporting their cargo to the translocation machinery in the peroxisomal membrane. A docking protein, Pex13p, for the yeast PTS1 (Pex5p) receptors has been found in S. cerevisiae and P. pastoris. It is an integral PMP and contains an SH3 domain (28Elgersma Y. Kwast L. Klein A. Voorn-Brouwer T. Van den Berg M. Metzig B. America T. Tabak H.F. Distel B. J. Cell Biol. 1996; 135: 97-109Crossref PubMed Scopus (183) Google Scholar, 37Erdmann R. Blobel G. J. Cell Biol. 1996; 135: 111-121Crossref PubMed Scopus (184) Google Scholar, 38Gould S.J. Kalish J.E. Morell J.C. Bjorkman J. Urquhart A.J. Crane D.I. J. Cell Biol. 1996; 135: 85-95Crossref PubMed Scopus (209) Google Scholar). Proteins involved in preventing entry of the receptors into the peroxisomal matrix or others playing a role in receptor recycling to the cytosol have been postulated, but no candidates have been identified yet (1Rachubinski R.A. Subramani S. How proteins penetrate peroxisomes, Cell. 1995; 83: 525-528Google Scholar).There appears to be a role for a 35-kDa zinc-binding PMP in the import of peroxisomal matrix proteins. The function of this protein and its relationship to several zinc-finger proteins known to reside in the peroxisomal membrane (listed in Ref. 23McNew J.A. Goodman J.M. Trends Biochem. Sci. 1996; 21: 54-58Abstract Full Text PDF PubMed Scopus (147) Google Scholar) are under investigation. 4S. Terlecky and S. Subramani, unpublished data.The genes for several integral PMPs involved in peroxisome biogenesis and/or import have been characterized, but it is not really clear whether they are affected mainly in peroxisome biogenesis or import, or both. The identities of PMPs constituting the translocation channel are likely to be derived from a combination of genetic and biochemical studies. Functional data are not yet available to show that the PMPs already characterized are directly involved in the peroxisomal protein translocation machinery.Folded State of Peroxisomal Matrix Proteins during Their ImportPTS1- and PTS2-containing proteins can be imported into peroxisomes in an oligomeric state that allows subunits lacking a PTS to be imported into peroxisomes in association with other subunits that do have a PTS (4Glover J.R. Andrews D.W. Rachubinski R.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10541-10545Crossref PubMed Scopus (244) Google Scholar, 5McNew J.A. Goodman J.M. J. Cell Biol. 1994; 127: 1245-1257Crossref PubMed Scopus (278) Google Scholar). Furthermore, gold particles conjugated to a peroxisomally targeted protein are imported into the organelle matrix (39Walton P.A. Hill P.E. Subramani S. Mol. Biol. Cell. 1995; 6: 675-683Crossref PubMed Scopus (210) Google Scholar). These results show that protein unfolding is not essential for the targeting of proteins to the peroxisomal matrix. Similar results are observed for glycosomes, which are related to peroxisomes (40Haüsler T. Stierhof Y. Wirtz E. Clayton C. J. Cell Biol. 1996; 132: 311-324Crossref PubMed Scopus (76) Google Scholar). The path taken by folded, oligomeric proteins across the peroxisomal membrane may either be through a gated pore or channel, or may involve an endocytosis-like mechanism at the peroxisomal membrane (2Subramani S. Curr. Opin. Cell Biol. 1996; 8: 513-518Crossref PubMed Scopus (41) Google Scholar). Protein unfolding requirements for PMPs have not been addressed.Biogenesis of Peroxisomal Membrane ProteinsIt is believed that PMPs are synthesized on free polysomes and then imported post-translationally to the peroxisome, without any involvement of the ER in the biogenesis. This fact has been documented for two mammalian proteins, PMP22 and PMP69/70 (7Fujiki Y. Rachubinski R.A. Lazarow P.B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7127-7131Crossref PubMed Scopus (146) Google