Endoplasmic Reticulum-directed Pex3p Routes to Peroxisomes and Restores Peroxisome Formation in a Saccharomyces cerevisiae pex3Δ Strain

Abstract

Recent studies on the sorting of peroxisomal membrane proteins challenge the long-standing model in which peroxisomes are considered to be autonomous organelles that multiply by growth and division. Here, we present data lending support to the idea that the endoplasmic reticulum (ER) is involved in sorting of the peroxisomal membrane protein Pex3p, a protein required early in peroxisome biogenesis. First, we show that the introduction of an artificial glycosylation site into the N terminus of Pex3p leads to partial N-linked core glycosylation, indicative of insertion into the ER membrane. Second, when FLAG-tagged Pex3p is equipped with an ER targeting signal, it can restore peroxisome formation in pex3Δ cells. Importantly, FLAG antibodies that specifically recognize the processed Pex3p show that the signal peptide of the fusion protein is efficiently cleaved off and that the processed protein localizes to peroxisomes. In contrast, a Pex3p construct in which cleavage of the signal peptide is blocked by a mutation localizes to the ER and the cytosol and cannot complement pex3Δ cells. Together, these results strongly suggest that ER-targeted Pex3p indeed routes via the ER to peroxisomes, and we hypothesize that this pathway is also used by endogenous Pex3p. Recent studies on the sorting of peroxisomal membrane proteins challenge the long-standing model in which peroxisomes are considered to be autonomous organelles that multiply by growth and division. Here, we present data lending support to the idea that the endoplasmic reticulum (ER) is involved in sorting of the peroxisomal membrane protein Pex3p, a protein required early in peroxisome biogenesis. First, we show that the introduction of an artificial glycosylation site into the N terminus of Pex3p leads to partial N-linked core glycosylation, indicative of insertion into the ER membrane. Second, when FLAG-tagged Pex3p is equipped with an ER targeting signal, it can restore peroxisome formation in pex3Δ cells. Importantly, FLAG antibodies that specifically recognize the processed Pex3p show that the signal peptide of the fusion protein is efficiently cleaved off and that the processed protein localizes to peroxisomes. In contrast, a Pex3p construct in which cleavage of the signal peptide is blocked by a mutation localizes to the ER and the cytosol and cannot complement pex3Δ cells. Together, these results strongly suggest that ER-targeted Pex3p indeed routes via the ER to peroxisomes, and we hypothesize that this pathway is also used by endogenous Pex3p. Peroxisomes are eukaryotic organelles surrounded by a single membrane. Depending on organism and cell type they can have different functions, two of which are well conserved, i.e. hydrogen peroxide decomposition and fatty acid β-oxidation. Peroxisome biogenesis is carried out by so-called peroxins encoded by PEX genes, 32 of which have been identified so far (1Distel 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. Kunau W.H. Lazarow P.B. Mannaerts G.P. Moser H.W. Osumi T. Rachubinski R.A. Roscher A. Subramani S. Tabak H.F. Tsukamoto T. Valle D. van der Klei I. van Veldhoven P.P. Veenhuis M. J. Cell Biol. 1996; 135: 1-3Crossref PubMed Scopus (314) Google Scholar, 2Vizeacoumar F.J. Torres-Guzman J.C. Bouard D. Aitchison J.D. Rachubinski R.A. Mol. Biol. Cell. 2004; 15: 665-677Crossref PubMed Scopus (97) Google Scholar). Peroxisomal matrix proteins are synthesized on free polyribosomes and posttranslationally imported into peroxisomes. Targeting of peroxisomal matrix proteins from the cytosol to peroxisomes is mediated by two types of peroxisomal targeting signals, PTS1 and PTS2, that are recognized by the corresponding mobile import receptors Pex5p and Pex7p, respectively (for review, see Ref. 3Purdue P.E. Lazarow P.B. Annu. Rev. Cell Dev. Biol. 2001; 17: 701-752Crossref PubMed Scopus (285) Google Scholar). Although trafficking of peroxisomal membrane proteins (PMPs) 2The abbreviations used are: PMP, peroxisomal membrane protein; SP, signal peptide; PTS, peroxisomal targeting signal; TRITC, tetramethylrhodamine isothiocyanate; ER, endoplasmic reticulum; Endo H, endoglycosidase H; GFP, green fluorescent protein; COP, coat protein. is less well understood, two observations indicate that distinct pathways exist for targeting of membrane and matrix proteins. First, in most pex mutants PMPs are correctly targeted to peroxisomal remnants (membrane structures lacking peroxisomal matrix proteins), whereas matrix proteins are found in the cytosol. The only pex mutants in which peroxisomal remnants are absent and PMPs are mislocalized and degraded are pex3 and pex19 in yeast and pex3, pex16, and pex19 in mammals (4Shimozawa N. Suzuki Y. Zhang Z. Imamura A. Tsukamoto T. Osumi T. Tateishi K. Okumoto K. Fujiki Y. Orii T. Barth P.G. Wanders R.J. Kondo N. Biochem. Biophys. Res. Commun. 1998; 243: 368-371Crossref PubMed Scopus (21) Google Scholar, 5South S.T. Gould S.J. J. Cell Biol. 1999; 144: 255-266Crossref PubMed Scopus (193) Google Scholar, 6Hettema E.H. Girzalsky W. van den Berg M. Erdmann R. Distel B. EMBO J. 2000; 19: 223-233Crossref PubMed Scopus (225) Google Scholar). This indicates that Pex3D, Pex16p, and Pex19p play important roles in peroxisomal membrane biogenesis in these organisms. Second, PMPs do not contain a PTS1 or PTS2 but, rather, possess a distinct membrane peroxisomal targeting signal consisting of a transmembrane domain in conjunction with a cluster of basic residues that frequently also contains a number of hydrophobic residues (7Dyer J.M. McNew J.A. Goodman J.M. J. Cell Biol. 1996; 133: 269-280Crossref PubMed Scopus (146) Google Scholar, 8Wang X. Unruh M.J. Goodman J.M. J. Biol. Chem. 2001; 276: 10897-10905Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 9Jones J.M. Morrell J.C. Gould S.J. J. Cell Biol. 2001; 153: 1141-1150Crossref PubMed Scopus (92) Google Scholar, 10Brosius U. Dehmel T. Gartner J. J. Biol. Chem. 2002; 277: 774-784Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 11Biermanns M. Gartner J. Biochem. Biophys. Res. Commun. 2001; 285: 649-655Crossref PubMed Scopus (28) Google Scholar). Recent studies of Gould and co-workers (9Jones J.M. Morrell J.C. Gould S.J. J. Cell Biol. 2001; 153: 1141-1150Crossref PubMed Scopus (92) Google Scholar, 12Jones J.M. Morrell J.C. Gould S.J. J. Cell Biol. 2004; 164: 57-67Crossref PubMed Scopus (194) Google Scholar) explain why Pex19p is essential for peroxisomal membrane biogenesis. These authors show that Pex19p functions as a chaperone and import receptor for PMPs, stabilizing newly synthesized PMPs and specifically interacting with the hydrophobic domains of the membrane peroxisomal targeting signals in these proteins. The fact that Pex19p is a predominantly cytosolic protein, with only a small percentage (<5%) localized to peroxisomes, suggests that it is a shuttling receptor, similar to Pex5p and Pex7p, binding its PMP cargo in the cytosol by to the peroxisome J. Biol. Chem. Full Text PDF PubMed Google Scholar, K. Girzalsky W. M. S. Kunau W.H. Erdmann R. Mol. Cell. Biol. 1998; PubMed Scopus Google Scholar, Y. N. S. N. M. K. Wanders R.J. Suzuki Y. Kondo N. Fujiki Y. U. S. 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This indicates that when Pex3p is the ER on it can the formation of peroxisomes in pex3Δ cells. that is indeed to peroxisomes via the ER, we the FLAG the and the N terminus of Pex3p The FLAG is recognized by two antibodies with different i.e. and The whereas the the FLAG only when it is present the N terminus of a fusion protein from which the signal peptide been by the signal in the This cleavage the of the FLAG which is essential for by the Subramani S. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). This be used to a been the ER, of its in the a we an to the cleavage site of a mutation to cleavage of the signal peptide J.D. G. S. J. Mol. Biol. 2004; PubMed Scopus Google be recognized by the not by the we also an FLAG-tagged Pex3p a signal peptide as a on the of the FLAG on Pex3p see and of pex3Δ or from the endogenous or the by the and antibodies The protein with the construct in a single in the and only recognized by the and that indeed an signal The protein from the construct also in a single recognized by antibodies and that the signal peptide of is Importantly, we a small in of the from the two the that the signal peptide of is and to and of the peptide cleavage to be as by the of the of the protein in the with the The of the in a in the of only the cleaved of the protein and that cleavage of the signal peptide of is of the to that the the FLAG only when it is present the N terminus of the by the signal in the the results of suggest that is efficiently processed by the ER signal that it is to the Peroxisome in a pex3 we is to restore peroxisome formation in a pex3Δ the of pex3Δ to on with as the in to the the of growth on that they contain peroxisomes. of the in to with indicative of a small of the FLAG on Pex3p Importantly, the construct with the to complement the growth of the pex3Δ results with the not an on peroxisome formation in we the protein and its with or from the endogenous a predominantly for with peroxisomes that import Morrell J.C. 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In contrast, a cytosolic in pex3Δ with the fusion protein from the endogenous a similar to that in pex3Δ with an and for peroxisomes. in this a of the FLAG on Pex3p is Although in peroxisomes peroxisomes in and to fluorescent these structures peroxisomes, we with antibodies the peroxisomal matrix In with and or and two types of peroxisomal the and similar to peroxisomes and the and indeed of of the fluorescent structures in B. found of organelles that or to the or to be with of the of in peroxisomes to be similar to that in that they contain a PTS2 import to peroxisomal structures and mislocalized to the cytosol Together, the of that a small of the FLAG ER-targeted is to formation of peroxisomes in pex3Δ the fusion protein is equipped with a signal in the ER and the that routes from the ER to peroxisomes, we the of the fusion protein by in pex3Δ cells. the construct is we used pex3Δ from the The construct is it is efficiently processed and the pex3Δ and data not In this we antibodies that specifically processed in with antibodies the peroxisomal matrix protein or the ER protein In processed to with or of In the processed and the protein only partial of These results can be by that processed a and is present in the ER and the peroxisomes, in by a of in the two in different cells. that these be to in of the in with a the protein to to the ER, whereas are required to a peroxisomal we pex3Δ and antibodies in with the These peroxisomes and in a of of with in the of the that it is present in the ER In to ER a that with that is also present in the These data support the that is targeted from the ER to peroxisomes on the of a The Pex3p is required early in peroxisome membrane and in so far of results in peroxisomal structures J. Veenhuis M. Kunau W.H. J. Cell Biol. PubMed Scopus Google Scholar, R.J. van der M. Kiel J.A. Cregg J.M. van der Klei Veenhuis M. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (146) Google Scholar, T. Veenhuis M. Subramani S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, N. Suzuki Y. Zhang Z. Imamura A. K. Fujiki Y. Kondo N. Mol. 2000; PubMed Scopus Google Scholar, R.A. Rachubinski R.A. Mol. Biol. Cell. PubMed Scopus Google Scholar). Here, we the of this essential and present that Pex3p to the ER the In a of and we an artificial site into the N terminus of Pex3p and that this construct a single core N-linked This indicates that Pex3p is into the ER membrane with its N terminus to the ER the model by J. Veenhuis M. Kunau W.H. J. Cell Biol. 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The introduction of a FLAG the to the construct indeed the ER to its These that and are proteins that can restore peroxisome formation in pex3Δ and and that is efficiently processed by the signal of the ER, of and that cleaved is localized in the ER and peroxisomes Together, these results strongly suggest that Pex3p equipped with an ER signal peptide indeed routes via the ER and show that ER of this protein results in the formation of peroxisomes in a pex3Δ of the pex3Δ is on the of a signal as by the to peroxisome formation of the a mutation in the signal peptide site and the is to complement pex3Δ is The fusion protein is targeted to the ER as with the ER it is not correctly processed is that the signal peptide in the ER sorting out of this observations have been for a signal peptide that cleavage by the signal M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that is also in the cytosol The cytosolic of be the of ER targeting be the of of processed Pex3p that been into the of to be these results support the that only the ER-targeted Pex3p is for peroxisome formation in pex3Δ cells. be a small of in the in most not be do these the of endogenous is that Pex3p contains that the protein to the proteins that are for the Pex3p not possess a signal and an ER targeting signal be S.T. Gould S.J. U. S. A. 2001; PubMed Scopus (50) Google Scholar) that of the ER or its not peroxisome biogenesis. This indeed suggests that Pex3p insertion into the ER not a signal peptide and of the insertion into the ER membrane is not it also been found for proteins J. 2002; PubMed Scopus Google Scholar, M. S. N. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google which are by a single transmembrane domain to the terminus and a cytosolic Pex3p, they an signal and also to be posttranslationally into the ER membrane. the of of ER import proteins and Pex3p, it be that they import that the processed is found in peroxisomes indicates the of a pathway in The growth on and the import of and proteins of pex3Δ indicate that ER-targeted Pex3p can efficiently restore the formation of peroxisomes. that Pex3p in these is the in peroxisome these results suggest that the pathway the by endogenous Pex3p. This is by results from studies in which endogenous of Pex3p localized to the ER into peroxisomes D. D. I. Tabak H.F. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). Pex3p from the ER to the peroxisomes is is to be mediated by of or not peroxisome biogenesis T. A. Tabak H.F. Distel B. J. Cell 2001; PubMed Google Scholar, S.T. X. Y. Gould S.J. J. Cell Biol. 2000; PubMed Scopus Google Scholar). 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T. Fujiki Y. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). data also suggest that Pex3p indeed the formation of peroxisomes the R.J. Veenhuis M. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) that of a Pex3p in pex3Δ in the of and that these in peroxisomes of Pex3p. In it in Y. that the formation of peroxisomes in pex3 mutants in with the ER R.A. Rachubinski R.A. Mol. Biol. Cell. PubMed Scopus Google Scholar). the of Pex3p trafficking to peroxisomes is different from that used by most Pex3p is the only protein so far identified that to peroxisomes of Pex19p J.M. Morrell J.C. Gould S.J. J. Cell Biol. 2004; 164: 57-67Crossref PubMed Scopus (194) Google Scholar) or for out that Pex3p is posttranslationally are to for and also van der for the and Erdmann for the Pex3p