Transcriptional Down-regulation of Peroxisome Numbers Affects Selective Peroxisome Degradation in Hansenula polymorpha
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m304029200
ISSN1083-351X
AutoresA LEAOHELDER, Arjen M. Krikken, Ida J. van der Klei, Jan A.K.W. Kiel, Marten Veenhuis,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoWe have isolated and characterized a novel transcription factor of Hansenula polymorpha that is involved in the regulation of peroxisomal protein levels. This protein, designated Mpp1p, belongs to the family of Zn(II)2Cys6 proteins. In cells deleted for the function of Mpp1p the levels of various proteins involved in peroxisome biogenesis (peroxins) and function (enzymes) are reduced compared with wild type or, in the case of the matrix protein dihydroxyacetone synthase, fully absent. Also, upon induction of mpp1 cells on methanol, the number of peroxisomes was strongly reduced relative to wild type cells and generally amounted to one organelle per cell. Remarkably, this single organelle was not susceptible to selective peroxisome degradation (pexophagy) and remained unaffected during exposure of methanol-induced cells to excess glucose conditions. We show that this mechanism is a general phenomenon in H. polymorpha in the case of cells that contain only a single peroxisome. We have isolated and characterized a novel transcription factor of Hansenula polymorpha that is involved in the regulation of peroxisomal protein levels. This protein, designated Mpp1p, belongs to the family of Zn(II)2Cys6 proteins. In cells deleted for the function of Mpp1p the levels of various proteins involved in peroxisome biogenesis (peroxins) and function (enzymes) are reduced compared with wild type or, in the case of the matrix protein dihydroxyacetone synthase, fully absent. Also, upon induction of mpp1 cells on methanol, the number of peroxisomes was strongly reduced relative to wild type cells and generally amounted to one organelle per cell. Remarkably, this single organelle was not susceptible to selective peroxisome degradation (pexophagy) and remained unaffected during exposure of methanol-induced cells to excess glucose conditions. We show that this mechanism is a general phenomenon in H. polymorpha in the case of cells that contain only a single peroxisome. Eukaryotic cells are thought to have evolved ∼1.5 billion years ago. The development of cell organelles allowed primitive eukaryotes to compartmentalize specific cellular functions. Concomitantly, genetic mechanisms that control the biogenesis and function of these compartments had to be developed. Obviously, the separate classes of organelles are characterized by their specific function, as in energy metabolism (mitochondrion), degradation processes (vacuole, lysosome), or protein transport (Golgi system, endoplasmatic reticulum). Among the organelles, peroxisomes are remarkable because of their highly versatile functions most of which are related to specific metabolic pathways in the organism in which they occur. This functional flexibility is not reflected in their morphology. The organelles are invariably very simple of construction and consist of a proteinaceous matrix, surrounded by a single membrane. Nevertheless, their function varies from the oxidation of very long chain fatty acids in man, germination of oil-bearing seed and photorespiration in green plants, to the metabolism of unusual carbon and/or nitrogen sources in fungi (1van den Bosch H. Schutgens R.B. Wanders R.J. Tager J.M. Annu. Rev. Biochem. 1992; 61: 157-197Crossref PubMed Scopus (737) Google Scholar). In the methylotrophic yeast Hansenula polymorpha peroxisomes are essential to support growth of cells on media containing methanol as the sole source of carbon and energy. Under these conditions many organelles that contain the key enzymes involved in methanol metabolism, alcohol oxidase (AO), 1The abbreviations used are: AO, alcohol oxidase; DHAS, dihydroxyacetone synthase; WT, wild type; ORF, open reading frame; GFP, green fluorescent protein. dihydroxyacetone synthase (DHAS), and catalase, develop in the cells. Conversely, when methanol-grown wild type (WT) cells are shifted to conditions in which the organelles are redundant for growth (e.g. glucose), they are rapidly and sequentially degraded by a process designated pexophagy (reviewed in Ref. 2Bellu A.R. Kiel J.A.K.W. Microsc. Res. Tech. 2003; 61: 161-170Crossref PubMed Scopus (56) Google Scholar). Morphological data suggest that in each cell generally a single (or few) small peroxisome(s) escape(s) the degradation process. The resistance of these organelles to degradation is thought to be of physiological advantage in that it allows the cells to quickly adapt to new environments that require new peroxisome functions. In the present work, we report the identification of a novel H. polymorpha transcription factor, Mpp1p, which is involved in the regulation of peroxisomal proteins. In H. polymorpha mpp1 cells, various peroxisomal matrix enzymes involved in methanol metabolism and proteins essential for peroxisome biogenesis (peroxins) are present at reduced levels. As a result, mpp1 cells cannot grow on methanol as the sole source of carbon and energy. Interestingly, methanol-induced mpp1 cells generally contained a single enlarged peroxisome. Remarkably, these single organelles are protected from selective degradation upon exposure of cells to excess glucose. Micro-organisms and Growth—The H. polymorpha strains used in this study are listed in Table I. H. polymorpha cells were grown at 37 °C in YPD media (1% yeast extract, 1% peptone, 1% glucose), selective minimal media containing 0.67% yeast nitrogen base without amino acids (Difco) supplemented with 1% glucose (YND) or 0.5% methanol (YNM) or mineral media (3van Dijken J.P. Otto R. Harder W. Arch. Microbiol. 1976; 111: 137-144Crossref PubMed Scopus (249) Google Scholar) supplemented with 0.5% glucose, 0.5% methanol, or 0.5% methanol + 0.1% glycerol. Whenever necessary, media were supplemented with 30 μg/ml leucine or 100 μg/ml zeocin. For growth on plates, 2% granulated agar was added to the media. For cloning purposes, Escherichia coli DH5α (Invitrogen) was used and grown at 37 °C in LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl), supplemented with 100 μg/liter ampicillin, 25 μg/liter kanamycin, or 25 μg/l zeocin when required.Table IH. polymorpha strains used in this studyStrainGenotype and characteristicsSourceNCYC495leu1.1 derivativeRef.44Gleeson M.A.G. Sudbery P.E. Yeast. 1988; 4: 293-303Crossref Scopus (121) Google ScholarNCYC495leu1.1 ura3 derivativeRef. 44Gleeson M.A.G. Sudbery P.E. Yeast. 1988; 4: 293-303Crossref Scopus (121) Google ScholarHF246NCYC495::(PAOX eGFP.SKL)1c, leu1.1Ref. 4van Dijk R. Faber K.N. Hammond A.T. Glick B.S. Veenhuis M. Kiel J.A.K.W. Mol. Genet. Genomics. 2001; 266: 646-656Crossref PubMed Scopus (43) Google Scholarmpp1—1HF246::[pREMI-Z], leu1.1, Mut—, zeoRThis studympp1—2HF246::[pREMI-Z], leu1.1, Mut—, zeoRRef. 4van Dijk R. Faber K.N. Hammond A.T. Glick B.S. Veenhuis M. Kiel J.A.K.W. Mol. Genet. Genomics. 2001; 266: 646-656Crossref PubMed Scopus (43) Google Scholarmpp1NCYC495 Δmpp1::HpURA3, leu1.1This studympp1.eGFP.SKLΔmpp1::(PAOXeGFP.SKL)1c, leu1.1, zeoRThis studyMPP1-eGFPNCYC495::MPP1-eGFP, leu1.1, zeoRThis study Open table in a new tab Gene Tagging Mutagenesis and Isolation of Mut - Mutants—The RALF (random integration of linear DNA fragments) method (4van Dijk R. Faber K.N. Hammond A.T. Glick B.S. Veenhuis M. Kiel J.A.K.W. Mol. Genet. Genomics. 2001; 266: 646-656Crossref PubMed Scopus (43) Google Scholar) was used to generate yeast mutants. H. polymorpha HF246 was transformed with BamHI-linearized pREMI-Z plasmid (Table II) in the presence of 1 unit of BamHI restriction enzyme. Transformants were initially selected on YPD plates supplemented with zeocin and subsequently replica-plated to YND and YNM plates, respectively. Colonies unable to grow on YNM plates (methanol utilization-defective, Mut-colonies) were further analyzed. Two Mut-mutants, designated mpp1-1 and mpp1-2 (previously designated ARJ-59; see Ref. 4van Dijk R. Faber K.N. Hammond A.T. Glick B.S. Veenhuis M. Kiel J.A.K.W. Mol. Genet. Genomics. 2001; 266: 646-656Crossref PubMed Scopus (43) Google Scholar), were studied further.Table IIPlasmids used in this studyPlasmidCharacteristicsSourcepBluescript II SK+Cloning vector, ampRStratagene, La Jolla, CApBSK-URA3pBluescript SK+ containing the 2.3-kb H. polymorpha URA3 fragment, ampRThis studypREMI-ZUsed for gene-tagging mutagenesis, zeoRRef. 4van Dijk R. Faber K.N. Hammond A.T. Glick B.S. Veenhuis M. Kiel J.A.K.W. Mol. Genet. Genomics. 2001; 266: 646-656Crossref PubMed Scopus (43) Google ScholarpANL7Rescued plasmid of mutant mpp1—1, obtained by digestion of chromosomal DNA with EcoRI followed by selfligation, zeoRThis studypANL15Rescued plasmid of mutant mpp1—1, obtained by digestion of chromosomal DNA with SphI followed by selfligation, zeoRThis studypANL17Plasmid containing the cassette for the deletion of the MPP1 gene, ampR, H. polymorpha URA3 geneThis studypREMI-59Rescued plasmid of mutant mpp1—2, obtained by digestion of chromosomal DNA with EcoRI followed by selfligation, zeoRThis studypANL22Rescued plasmid of mutant mpp1—2, obtained by digestion of chromosomal DNA with SphI followed by selfligation, zeoRThis studypANL26E. coli/H. polymorpha shuttle plasmid containing the uninterrupted MPP1 gene, ampR, S. cerevisiae LEU2 gene, HARS1This studypANL29pHIPZ4 containing eGFP.SKL, zeoR, ampRThis studypANL31pBluescript derivative containing the eGFP gene without a startcodon, zeoR, ampRThis studypANL32pANL31 with a 748-bp fragment containing the 3′ end of the MPP1 gene fused in-frame to the eGFP gene, zeoR, ampRThis studypFEM34Plasmid containing the PAOX.eGFP.SKL cassette and the kanR, S. cerevisiae LEU2 geneRef. 7Faber K.N. van Dijk R. Keizer-Gunnink I. Koek A. van der Klei I.J. Veenhuis M. Biochim. Biophys. Acta. 2002; 1591: 157-162Crossref PubMed Scopus (35) Google ScholarpHIPZ4Integrative plasmid for H. polymorpha, contains the H. polymorpha AOX promoter, zeoR, ampRRef. 8Salomons F.A. Kiel J.A.K.W. Faber K.N. Veenhuis M. van der Klei I.J. J. Biol. Chem. 2000; 275: 12603-12611Abstract Full Text Full Text PDF PubMed Scopus (72) Google ScholarpX3-PPAOXβlacPlasmid expressing the β-lactamase gene under control of the AOX promoterRef. 19Waterham H.R. Titorenko V.I. Haima P. Cregg J.M. Harder W. Veenhuis M. J. Cell Biol. 1994; 127: 737-749Crossref PubMed Scopus (174) Google ScholarpX3-PPEX3βlacPlasmid expressing the β-lactamase gene under control of the PEX3 promoterRef. 23Baerends R.J.S. Hilbrands R.E. van der Heide M. Faber K.N. Reuvekamp P.T. Kiel J.A.K.W. Cregg J.M. Van der Klei I.J. Veenhuis M. J. Biol. Chem. 1996; 271: 8887-8894Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar Open table in a new tab Cloning of the MPP1 Gene—To identify the gene(s) disrupted by pREMI-Z in mutants mpp1-1 and mpp1-2, the chromosomal DNA of the cells was digested with either EcoRI or SphI, self-ligated, and transformed to E. coli, giving rise to the following plasmids: pANL7, pANL15, pREMI-59, and pANL22 (Table II). The genomic regions of pANL7 and pREMI-59 were initially sequenced using vector-based primers (4van Dijk R. Faber K.N. Hammond A.T. Glick B.S. Veenhuis M. Kiel J.A.K.W. Mol. Genet. Genomics. 2001; 266: 646-656Crossref PubMed Scopus (43) Google Scholar). Sequence analysis showed that the pREMI-Z vector had integrated in mutants mpp1-1 and mpp1-2 at two different locations in the same gene that was designated MPP1. Subsequently, the entire nucleotide sequence of the MPP1 gene was determined by primer walking on the rescued pREMI-Z plasmids. The nucleotide sequence of the MPP1 gene was deposited in GenBank™ (accession number AY190521). To clone the MPP1 open reading frame, mutant mpp1-1 was transformed with a H. polymorpha genomic library constructed in the pYT3 vector (5Tan X. Waterham H.R. Veenhuis M. Cregg J.M. J. Cell Biol. 1995; 128: 307-319Crossref PubMed Scopus (119) Google Scholar). Leucine prototrophic transformants were screened on YNM plates for the ability to grow on methanol. From a complementing plasmid, a sub-clone containing a 3.4-kb PstI-NheI fragment with the entire MPP1 gene was obtained, designated pANL26, that was used for complementation studies. Construction of an H. polymorpha MPP1 Null Mutant—A strain deleted for MPP1 was constructed by replacing the region of MPP1 comprising nucleotides +1 to +1042 by an auxotrophic marker. To this end, a deletion cassette was constructed as follows. First, two DNA fragments comprising the regions -816 to -1 and +1042 to +1841 of the MPP1 genomic region were obtained by PCR, using primers MPP1del-1 + MPP1del-2 and MPP1del-3 + MPP1del-4, respectively (see Table III). After restriction with NotI + BglII and PstI + Asp718I, respectively, the resulting fragments were inserted upstream and downstream of the H. polymorpha URA3 gene (6Merckelbach A. Godecke S. Janowicz Z.A. Hollenberg C.P. Appl. Microbiol. Biotechnol. 1993; 40: 361-364Crossref PubMed Scopus (44) Google Scholar) in pBSK-URA3. From the resulting plasmid, designated pANL17, a 2650-bp BamHI-PvuI fragment was used to transform H. polymorpha NCYC495 leu1.1 ura3. Uracil prototrophic transformants were selected by their inability to grow on YNM plates. Proper deletion of MPP1 was confirmed by Southern blotting (data not shown). The resulting strain was designated mpp1.Table IIIPrimers used in this studyNameSequenceMPP1-del15′-AGAGAGAGGCGGCCGCGGCTATAGACGCTC G-3′NotIMPP1-del25′-AGAGATCTTTTGGAGCAAAACCTGAGC-3′Bg1IIMPP1-del35′-AGCTGCAGTTGCCTCCCGACACCTTG-3′PstIMPP1-del45′-AGAGGTACCACCGATGATCTGCTTGGC-3′Asp718IMPP1-Ori25′-GACACCTTGAGAAAGTCAGATC-3′MPP1w/oStop5′-AGAGGATCCGCACTCGCGTTTCCAG-3′BamHI Open table in a new tab To enable visualization of peroxisomes by fluorescence microscopy, the eGFP.SKL reporter gene was introduced in the resulting mpp1 strain. First, we constructed a H. polymorpha integrative plasmid containing the PAOX .eGFP.SKL cassette and the zeocin-resistance gene by inserting the 1.2-kb SphI-SalI fragment of pFEM34 (7Faber K.N. van Dijk R. Keizer-Gunnink I. Koek A. van der Klei I.J. Veenhuis M. Biochim. Biophys. Acta. 2002; 1591: 157-162Crossref PubMed Scopus (35) Google Scholar) in pHIPZ4 (8Salomons F.A. Kiel J.A.K.W. Faber K.N. Veenhuis M. van der Klei I.J. J. Biol. Chem. 2000; 275: 12603-12611Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Subsequently, the resulting plasmid, designated pANL29, was linearized with SphI and integrated in the mpp1 genome. Zeocin-resistant transformants were analyzed for correct integration in the PAOX region by Southern blotting (data not shown). A strain containing a single copy of the integrated plasmid, designated mpp1.eGFP.SKL, was used for further studies. Construction of a Strain Expressing an MPP1.eGFP Fusion Gene—To enable replacement of the genomic MPP1 gene by a MPP1.eGFP fusion gene, we first constructed plasmid pANL31. This plasmid is based on the pBluescript vector and contains an eGFP gene, lacking its start codon, and the zeocin resistance cassette. Subsequently, a 749bp Hin-dIII-BamHI PCR fragment containing the 3′ end of the MPP1 gene lacking its stop codon (comprising the region +1306 to +2052 of MPP1), obtained using primers MPP1-Ori2 and MPP1w/oStop (Table III), was inserted in HindIII/BglII-digested pANL31. The resulting plasmid, designated pANL32, was linearized with EcoRI in the MPP1 region and transformed to WT H. polymorpha NCYC495 leu 1.1. Zeocin-resistant colonies were analyzed by Southern blotting to confirm correct integration in the MPP1 region (data not shown). Miscellaneous DNA Techniques—Plasmids and primers used in this study are listed in Tables II and III, respectively. All DNA manipulations were carried out according to standard techniques (9Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). H. polymorpha cells were transformed by electroporation (10Faber K.N. Haima P. Harder W. Veenhuis M. AB G. Curr. Genet. 1994; 25: 305-310Crossref PubMed Scopus (218) Google Scholar). DNA modifying enzymes were used as recommended by the supplier (Roche Applied Science). Pwo polymerase was used for preparative PCR. The ECL direct nucleic acid labeling and detection system (Amersham Biosciences) was used for Southern blot analysis. Oligonucleotides were synthesized by Invitrogen. DNA sequencing reactions were performed at BaseClear (Leiden, The Netherlands) using a LiCor automated DNA sequencer and dye primer chemistry (LiCor, Lincoln, NB). For DNA sequence analysis, the Clone Manager 5 program (Scientific and Educational Software, Durham, NC) was used. The BLASTP algorithm (11Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59915) Google Scholar) was used to screen databases at the National Center for Biotechnology Information (Bethesda, MD). The ClustalX program was used to align protein sequences (12Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35486) Google Scholar), the GeneDoc program was used to display the aligned protein sequences, and the ScanProsite program (13Gattiker A. Gasteiger E. Bairoch A. Appl. Bioinform. 2002; 1: 107-108PubMed Google Scholar) was used to scan protein sequences for profiles and patterns of the PROSITE data base (14Falquet L. Pagni M. Bucher P. Hulo N. Sigrist C.J. Hofmann K. Bairoch A. Nucleic Acids Res. 2002; 30: 235-238Crossref PubMed Scopus (902) Google Scholar). Biochemical Assays—Crude cell extracts were prepared as described (15Baerends R.J.S. Faber K.N. Kram A.M. Kiel J.A.K.W. van der Klei I.J. Veenhuis M. J. Biol. Chem. 2000; 275: 9986-9995Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). SDS-PAGE (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207200) Google Scholar) and Western blot analysis (17Kyhse-Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Crossref PubMed Scopus (2158) Google Scholar) were performed by established methods. The degradation of peroxisomes in batch cultures of H. polymorpha was determined as described (18Titorenko V.I. Keizer I. Harder W. Veenhuis M. J. Bacteriol. 1995; 177: 357-363Crossref PubMed Scopus (93) Google Scholar). Relative AO levels were determined by densitometric scanning of Western blots decorated with specific antibodies against AO. The decrease in AO levels during peroxisome degradation is expressed as a percentage of the initial value, which is arbitrarily set to 100%. β-Lactamase activities were assayed by established methods (19Waterham H.R. Titorenko V.I. Haima P. Cregg J.M. Harder W. Veenhuis M. J. Cell Biol. 1994; 127: 737-749Crossref PubMed Scopus (174) Google Scholar). Morphological Analysis—Intact cells were prepared for electron microscopy and immunocytochemistry as described previously (19Waterham H.R. Titorenko V.I. Haima P. Cregg J.M. Harder W. Veenhuis M. J. Cell Biol. 1994; 127: 737-749Crossref PubMed Scopus (174) Google Scholar). Fluorescence microscopy studies were performed using a Zeiss Axioskop microscope (Carl Zeiss, Göttingen, Germany). Nuclear staining was performed as follows: 20-40 OD660 units of cells were harvested by centrifugation, resuspended in 1 ml of fresh medium supplemented with 40 μl of a 2.7 mg/ml Hoechst 33258 stock solution, and incubated at 37 °C for 45 min prior to analysis. Mpp1p Is a Member of the Zn(II) 2 Cys 6 Cluster Protein Family and Is Induced during Growth of H. polymorpha on Methanol— From a collection of 5,000 RALF transformants, mutants were selected that were impaired in growth on methanol as sole carbon source (Mut-phenotype). Two mutants, designated mpp1-1 and mpp1-2, were identified of identical morphological phenotype. Characteristically, upon growth in glycerol/methanol-containing media, conditions that lead to a strong peroxisome development in WT cells, generally a single peroxisome was observed in the vast majority of the mpp1-1 and mpp1-2 cells (Fig. 1). Sequencing of the plasmids recovered from these mutants revealed that the flanking regions were overlapping. Hence, both were apparently disturbed in the function of the same gene, termed MPP1 (methylotrophic peroxisomal protein regulator 1). Further sequencing of the plasmids recovered from mutants mpp1-1 and mpp1-2 allowed the determination of the remainder of the MPP1 open reading frame (ORF). Sequence analysis indicated that in case of mutant mpp1-1, the integration of the pREMI-Z plasmid had occurred 456 bp downstream from the initiation of the MPP1 ORF. In mutant mpp1-2, pREMI-Z had inserted at the promoter region of the same ORF (nucleotide -195). The sequence upstream the 5′ region of this ORF comprised the 3′ end of the H. polymorpha dihydroxyacetone synthase gene (DAS; see Fig. 2A).Fig. 2The H. polymorpha MPP1 gene. A, schematic representation of the genomic region comprising the H. polymorpha MPP1 gene. The strategy to disrupt MPP1 by homologous recombination is presented. The integration sites of pREMI-Z in the mpp1-1 and mpp1-2 mutants are also indicated. Only relevant restriction sites are shown. B, alignment of the N termini of Mpp1p and other characteristic yeast Zn(II)2Cys6 proteins. The N-terminal amino acid sequences of S. cerevisiae Gal4p (SwissProt accession number P04386; amino acids 1-58), Pip2p (SwissProt accession number P52960; amino acids 1-72), and Pdr3p (SwissProt accession number P33200; amino acids 1-61) were aligned with that of H. polymorpha Mpp1p (amino acids 1-75) using the ClustalX program. The one-letter code is shown. Gaps were introduced to maximize the similarity. Residues that are similar in all four proteins are shaded black, those that are similar in three of the proteins are shaded dark gray, and those that are similar in two of the four proteins are shaded light gray.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The MPP1 gene encodes a protein of 684 amino acids. A BLASTP search revealed that the N-terminal region of Mpp1p is similar to that of many DNA-binding proteins. However, a true homologue was not found in the available databases. Further analysis using the ScanPROSITE program revealed that Mpp1p is a putative member of the Zn(II)2Cys6 family of transcription factors. These transcription regulators are exclusively detected in fungi and contain a well conserved DNA binding domain (20Schjerling P. Holmberg S. Nucleic Acids Res. 1996; 24: 4599-4607Crossref PubMed Scopus (223) Google Scholar). This domain consists of a cystein-rich motif (CysX 2CysX 6CysX 6CysX 2CysX 6Cys) that complexes two Zn2+ ions and in most cases recognizes a pair of 5′-CGG-3′ triplets in the promoters of target genes (20Schjerling P. Holmberg S. Nucleic Acids Res. 1996; 24: 4599-4607Crossref PubMed Scopus (223) Google Scholar, 21Pan T. Coleman J.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2077-2081Crossref PubMed Scopus (162) Google Scholar). A primary sequence comparison of the region comprising the DNA binding domain of characteristic Zn(II)2Cys6 proteins of Saccharomyces cerevisiae and the putative DNA binding domain of Mpp1p is depicted in Fig. 2B. The subcellular location of Mpp1p in H. polymorpha was studied in a strain in which the endogenous MPP1 gene was replaced by a MPP1.eGFP fusion gene. The presence of the C-terminal tag most likely did not interfere with Mpp1p function, because the resulting strain, MPP1.eGFP, grew normally on glucose and methanol (data not shown). In MPP1.eGFP cells grown on glucose, GFP fluorescence was invariably undetectable, independent of the growth stage. However, upon a shift of glucose-grown cell to fresh methanol-containing media, GFP fluorescence was readily observed as a single dot at all growth phases. This spot was observed in the same region of the cells as Hoechst 33258, which is a nuclear stain. These findings therefore indicate that Mpp1p is associated with the nucleus (Fig. 3). Cells of the MPP1 Deletion Strain Generally Contain a Single Peroxisome—For construction of a MPP1 deletion strain, we replaced a 1042-bp fragment from the MPP1 open reading frame by the H. polymorpha URA3 gene (Fig. 2A). Cells of the mpp1 strain and the original RALF mutants, mpp1-1 and mpp1-2, showed identical phenotypes. Mpp1 cells were unable to grow on methanol as sole carbon source and, when grown on glycerol/methanol mixtures, characteristically contained a single peroxisome (Fig. 4B). Reintroduction of the MPP1 gene from an autonomously replicating plasmid (pANL26) restored normal growth of mpp1 cells on methanol, as well as normal peroxisome proliferation (Fig. 4C). In crude extracts of glycerol/methanol-grown WT and mpp1 cells, the levels of the peroxisomal matrix protein AO were strongly reduced, whereas DHAS, another peroxisomal matrix enzyme, could not be detected (Fig. 5A). However, catalase was present at approximately WT levels. Also the levels of malate synthase, a peroxisomal key enzyme of glyoxylate (C2) metabolism (22Bruinenberg P.G. Blaauw M. Kazemier B. AB G. Yeast. 1990; 6: 245-254Crossref PubMed Scopus (17) Google Scholar), did not change significantly under the conditions tested (Fig. 5A). In addition, the level of various peroxins that play a role in peroxisome formation was analyzed. All were present at significantly reduced levels (Pex3p, Pex5p, Pex10p) with the exception of Pex14p (Fig. 5B). Cytosolic alcohol dehydrogenase levels were normal. To test whether the observed lower protein levels were because of decreased expression or increased protein degradation, we analyzed the promoter activities of the AOX (PAOX) and PEX3 (PPEX3) genes in WT and mpp1 cells using β-lactamase as a reporter (19Waterham H.R. Titorenko V.I. Haima P. Cregg J.M. Harder W. Veenhuis M. J. Cell Biol. 1994; 127: 737-749Crossref PubMed Scopus (174) Google Scholar, 23Baerends R.J.S. Hilbrands R.E. van der Heide M. Faber K.N. Reuvekamp P.T. Kiel J.A.K.W. Cregg J.M. Van der Klei I.J. Veenhuis M. J. Biol. Chem. 1996; 271: 8887-8894Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). As shown in Fig. 5C, the activities of both promoters are significantly reduced in mpp1 cells compared with WT controls. Despite the low peroxisome numbers we did not observe a defect in matrix protein import in mpp1 cells. GFP.SKL was solely observed in spots (compare Fig. 4B), and immunocyto-chemistry revealed that anti-alcohol oxidase and anti-catalase-dependent-specific labeling was confined to the peroxisomal profiles (Fig. 4D), indicating that these proteins were incorporated in their correct target organelle. As expected, using antidihydroxyacetone synthase antibodies, specific labeling was not detected (not shown). Single Peroxisomes in H. polymorpha Cells Are Not Susceptible to Glucose-induced Selective Peroxisome Degradation (Pexophagy)—In methanol-grown WT H. polymorpha cells peroxisomes are rapidly degraded when they have become redundant for growth (24Veenhuis M. Douma A. Harder W. Osumi M. Arch. Microbiol. 1983; 134: 193-203Crossref PubMed Scopus (156) Google Scholar). To analyze the fate of the single organelles in mpp1 cells, glycerol/methanol-grown cells of this strain were exposed to excess glucose conditions. In the first 2 h after the shift of cells, no significant AO protein degradation had occurred in mpp1 cells, as judged from Western blots (Fig. 6, A and B). In mpp1.eGFP.SKL cells, fluorescence microscopy studies failed to demonstrate the uptake of the fluorescent reporter protein in vacuoles, a phenomenon that was readily observed in WT controls (Fig. 6C). Also, electron microscopically we were never able to detect peroxisome sequestration, the typical initial event of pexophagy in H. polymorpha (not shown). To further substantiate the possibility that also in WT cells specific organelles escape the pexophagy process, we have grown H. polymorpha WT cells on methanol to a phase that they generally contain only one or two organelles per cell (early exponential growth; see Ref. 25Veenhuis M. Keizer I. Harder W. Arch. Microbiol. 1979; 120: 167-175Crossref Scopus (87) Google Scholar) and studied pexophagy relative to cells that were in the mid-exponential growth phase and contained several peroxisomes. The results clearly show that the single peroxisome present in early exponential WT cells is not degraded within a period of 4 h after exposure of the cells to excess glucose (Fig. 7C). In contrast, in mid-exponential cells a rapid reduction of peroxisome numbers was observed. Nevertheless, in these cells generally a single fluorescent spot remained indicating that not all peroxisomes were degraded (Fig. 7C). Remarkably, the morphological phenotypes of early and mid-exponential WT cells after 4 h of exposure of cells to glucose was indistinguishable in that they generally contained a single fluorescent spot. Most likely, solely small organelles remain unaffected as was evident after careful electron microscopical observations (Fig. 4E). These observations were confirmed biochemically by Western blot analysis of samples taken from the same cultures using specific antibodies against AO (Fig. 7, A and B). When WT cells in the early exponential growth phase were subjected to pexophagy conditions, AO protein levels only slightly decreased during the first hour after the shift. However, at later time points the AO levels remained constant, indicating that the peroxisomes in these cells were not degraded. The initial decrease most likely is because of the presence of a minor fraction of the cells that contains more than one peroxisome at the time of the shift. Mid-exponentially grown cells showed the continuous decrease in AO pro
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