Sue1p Is Required for Degradation of Labile Forms of Altered Cytochromes c in Yeast Mitochondria
2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês
10.1074/jbc.m403742200
ISSN1083-351X
Autores Tópico(s)ATP Synthase and ATPases Research
ResumoPrevious studies on certain altered holo-isocytochromes c revealed a ρ–-dependent degradation (RDD) phenotype, in which certain altered holo-iso-1-cytochromes c are at normal or nearly normal levels in ρ+ strains, but are at low levels or absent in ρ– strains, although wild-type holo-iso-1-cytochrome c is present at normal levels in both ρ+ and related ρ– strains. The diminished levels of altered holo-iso-1-cytochrome c are due to the rapid degradation that is carried out by a novel proteolytic pathway in the IMS of mitochondria. SUE1, a nuclear gene that encodes a mitochondrial protein, was identified with a genetic screen for mutants that diminish RDD. The levels of RDD and certain other types of altered holo-iso-1-cytochrome c were elevated in ρ– sue1 strains. Also, ρ+ sue1 strains containing certain altered holo-iso-1-cytochromes c grew better on non-fermentable carbon sources than the corresponding ρ+ SUE1 strains. These results indicate that Sue1p may play an important role in the degradation of abnormal holo-iso-1-cytochrome c in the mitochondria. Previous studies on certain altered holo-isocytochromes c revealed a ρ–-dependent degradation (RDD) phenotype, in which certain altered holo-iso-1-cytochromes c are at normal or nearly normal levels in ρ+ strains, but are at low levels or absent in ρ– strains, although wild-type holo-iso-1-cytochrome c is present at normal levels in both ρ+ and related ρ– strains. The diminished levels of altered holo-iso-1-cytochrome c are due to the rapid degradation that is carried out by a novel proteolytic pathway in the IMS of mitochondria. SUE1, a nuclear gene that encodes a mitochondrial protein, was identified with a genetic screen for mutants that diminish RDD. The levels of RDD and certain other types of altered holo-iso-1-cytochrome c were elevated in ρ– sue1 strains. Also, ρ+ sue1 strains containing certain altered holo-iso-1-cytochromes c grew better on non-fermentable carbon sources than the corresponding ρ+ SUE1 strains. These results indicate that Sue1p may play an important role in the degradation of abnormal holo-iso-1-cytochrome c in the mitochondria. Intracellular proteolysis plays an important role in maintaining the integrity of the proper folded state of proteins. It ensures removal of damaged and misfolded polypeptides because they are prone to aggregation. A basic mechanism for control of protein degradation is compartmentalization (1Baumeister W. Walz J. Zuhl F. Seemuller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1310) Google Scholar). In eukaryotic cells, proteases have been detected in four compartments: the cytoplasm, nucleus, lysosome, and mitochondrion. The mitochondria have various subcompartments that possess ATP-dependent proteases as a quality control system for selectively removing unassembled or misfolded polypeptides. Several ATP-dependent proteases such as the Pim1 protease in the matrix or AAA (ATPases associated with a variety of cellular activities) proteases in the inner membrane of mitochondria have been identified (2Goldberg A.L. Eur. J. Biochem. 1992; 203: 9-23Crossref PubMed Scopus (415) Google Scholar, 3Goldberg A.L. Akopian T.N. Kisselev A.F. Lee D.H. Rohrwild M. Biol. Chem. 1997; 378: 131-140PubMed Google Scholar, 4Porankiewicz J. Wang J. Clarke A.K. Mol. Microbiol. 1999; 32: 449-458Crossref PubMed Scopus (182) Google Scholar, 5Käser M. Langer T. Cell. Dev. Biol. 2000; 11: 181-190Crossref PubMed Scopus (73) Google Scholar). Additional proteolytic pathways may exist in the other two subcompartments of mitochondria: the intermembrane space (IMS) 1The abbreviations used are: IMS, intermembrane space; holo-1, holo-iso-1-cytochrome(s) c; holo-2, holo-iso-2-cytochrome(s) c; iso-1, iso-1-cytochrome(s) c; iso-2, iso-2-cytochrome(s) c; RDD, ρ–-dependent degradation; LDD, labile dependent degradation; ADD, amphipathic dependent degradation; GFP, green fluorescent protein; TBS, Tris-buffered saline; Nfs–, diminished or lack of growth on media containing non-fermentable carbon sources as the sole source of energy, such as glycerol and ethanol. and outer membrane. The existence of an ATP-dependent proteolytic activity in the mitochondrial IMS in mammals has been reported, although the ATP-dependent protease so far has not been identified (6Sitte N. Dubiel W. Kloetzel P.M. J. Biochem. (Tokyo). 1998; 123: 408-415Crossref PubMed Scopus (12) Google Scholar, 7Granot Z. Geiss-Friedlander R. Melamed-Book N. Eimerl S. Timberg R. Weiss A.M. Hales K.H. Hales D.B. Stocco D.M. Orly J. Mol. Endocrinol. 2003; 17: 2461-2476Crossref PubMed Scopus (78) Google Scholar). We report herein a proteolytic pathway in the IMS of mitochondria acting on certain altered holo-iso-1-cytochromes c (holo-1) of the yeast Saccharomyces cerevisiae. S. cerevisiae contains two forms of cytochrome c, iso-1-cytochrome c (iso-1) and iso-2-cytochrome c (iso-2), which are encoded by the nuclear genes CYC1 and CYC7, which normally compose 95 and 5% of total cytochrome c, respectively, in aerobically grown, derepressed cells (8Sherman F. Taber H. Campbell W. J. Mol. Biol. 1965; 13: 21-39Crossref PubMed Scopus (75) Google Scholar) and which are 80% identical. The isocytochromes c are synthesized in the cytosol as apocytochromes c and subsequently imported into mitochondria. Heme is covalently attached to the apocytochromes c by cytochrome c heme lyase, which is encoded by the gene CYC3, resulting in the formation of the mature holocytochromes c (9Dumont M.E. Ernst J.F. Hampsey D.M. Sherman F. EMBO J. 1987; 6: 235-241Crossref PubMed Scopus (165) Google Scholar). Import of the apocytochromes c is dependent on the action of cytochrome c heme lyase, and cyc3-Δ mutants lacking cytochrome c heme lyase accumulate apocytochromes c in the cytosol (10Dumont M. Ernst J.F. Sherman F. J. Biol. Chem. 1988; 263: 15928-15937Abstract Full Text PDF PubMed Google Scholar). However, apo-iso-1-cytochrome c is not detected in cyc3-Δ mutants, whereas apo-iso-2-cytochrome c is present at the corresponding level of holo-iso-2-cytochrome c (holo-2) in related normal CYC3 strains (11Matner R.R. Sherman F. J. Biol. Chem. 1982; 257: 9811-9821Abstract Full Text PDF PubMed Google Scholar). Dumont et al. (12Dumont M.E. Mathews A.J. Nall B.T. Baim S.B. Eustice D.C. Sherman F. J. Biol. Chem. 1990; 265: 2733-2739Abstract Full Text PDF PubMed Google Scholar) demonstrated with pulse-chase experiments that unimported apo-iso-1-cytochrome c is rapidly degraded, and Pearce and Sherman (13Pearce D.A. Sherman F. J. Biol. Chem. 1997; 272: 31829-31836Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) demonstrated that this apo-iso-1-cytochrome c degradation requires functional proteasomes and is mediated by the ubiquitin-dependent pathway. In contrast, holo-1 and holo-2 are highly stable. We have been identifying and characterizing mitochondrial protein degradation systems by investigating mutant forms of iso-1 that are rapidly degraded and by determining mutations of other genes that diminish the degradation. Downie et al. (14Downie J.A. Stewart J.W. Brockman N. Schweinruber A.M. Sherman F. J. Mol. Biol. 1977; 113: 369-386Crossref PubMed Scopus (32) Google Scholar) observed that certain mutated holo-2 are at normal or nearly normal levels in ρ+ strains, but are absent in ρ– strains, although wild-type isocytochromes c are present at normal levels in both ρ+ and related ρ– strains. Subsequently, Pearce and Sherman (15Pearce D.A. Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3735-3739Crossref PubMed Scopus (20) Google Scholar, 17Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google Scholar) generated equivalent mutants of holo-1 and showed that amino acid replacements at several positions of holo-1 diminish the levels of holo-1, preferentially in ρ– strains. This phenomenon is referred to as the ρ–-dependent degradation (RDD) phenotype. The iso-1 that shows RDD is referred to as RDD iso-1. In addition, pulse-chase experiments with a variety of altered forms of cytochrome c revealed two other degradation pathways acting on other altered forms of cytochrome c, labile dependent degradation (LDD) and amphipathic dependent degradation (ADD). LDD, exemplified by Gly6 replacements, does not have diminished levels in ρ– strains (15Pearce D.A. Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3735-3739Crossref PubMed Scopus (20) Google Scholar, 17Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google Scholar). ADD, containing amphipathic structures in the N-terminal region, also does not have diminished levels in ρ– strains. 2X. Chen, R. P. Moerschell, D. A. Pearce, D. D. Ramanan, and F. Sherman, unpublished data. The cytochromes c degraded by these pathways are designated LDD and ADD cytochromes c, respectively. The introduction of the global suppressor N52I (18Das G. Hickey D.R. McLendon D. McLendon G. Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 496-499Crossref PubMed Scopus (94) Google Scholar) into both RDD and LDD holo-1 partially restores the levels of RDD holo-1 in ρ– strains (17Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google Scholar) and gives rise to better growth on a non-fermentable carbon source for LDD holo-1 in ρ+ strains (17Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google Scholar). Moreover, Pearce and Sherman (15Pearce D.A. Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3735-3739Crossref PubMed Scopus (20) Google Scholar) demonstrated that the absence of either cytochrome aa3 or cytochrome c1, the physiological partner of cytochrome c in the IMS of mitochondria, causes increased sensitivity of RDD holo-1 to degradation. These observations suggest that the degradation of RDD and LDD holo-1 may occur in the IMS of mitochondria. All known ATP-dependent mitochondrial proteases (Yme1p, Afg3p, and Rca1p) are not involved in the degradation of T78I RDD holo-1 (15Pearce D.A. Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3735-3739Crossref PubMed Scopus (20) Google Scholar). Therefore, the protease responsible for the degradation of RDD holo-1 in the IMS of mitochondria could be a novel protease. We investigated this novel protease system genetically by using strain cyc1-1388 with T78S RDD holo-1 for detecting mutants with diminished degradation. Strain cyc1-1388 was mutagenized, and colonies containing increased levels of cytochrome c were detected by the benzidine staining procedure, in which the color intensity is proportional to the level of holo-1 (19Sherman F. Stewart J.W. Parker J.H. Inhaber E. Shipman N.A. J. Biol. Chem. 1968; 243: 5446-5456Abstract Full Text PDF PubMed Google Scholar). The levels of cytochrome c were subsequently quantified by low temperature spectroscopic examination of intact cells. High levels of RDD holo-1 in these ρ– mutants could be due to the direct or indirect diminution of a protease. One recessive nuclear mutation was characterized and used to identify SUE1. The degradation of three classes of altered holo-1 (RDD, LDD, and ADD holo-1) was investigated in sue1-Δ strains. Elevated levels of altered holo-1 (T78S RDD, G6I LDD, and ADD holo-1) were observed in ρ– sue1-Δ strains. Furthermore, better growth was observed in ρ+ sue1-Δ strains containing G6I LDD or ADD holo-1. These results indicate that Sue1p plays an important role in the degradation of some labile forms of altered holo-1 in the mitochondria. Genetic Nomenclature—CYC1 and CYC7 encode iso-1 and iso-2, respectively, the two isozymes of cytochrome c in the yeast S. cerevisiae. Dominant alleles are denoted with uppercase letters, and recessive alleles with lowercase letters. CYC1 refers to the wild-type allele, whereas the mutant allele of the CYC1 gene is designated cyc1-X, such as the cyc1-1388 mutant allele. The cyc7-67 allele corresponds to a partial deletion that results in the loss of iso-2. SUE1, identified in this study, refers to the wild-type gene that encodes a mitochondrial protein. The sue1-Δ mutant allele completely lacks Sue1p, and the sue1-2 mutant allele contains a combined nonsense and frameshift mutation at position 9, resulting in a nonfunctional Sue1p. The yeast strains used in this study are listed in Table I, and the cyc1 alleles used in this study are listed in Table II. Iso-1 encoded by, for example, cyc1-1388 is designated Cyc1-1388p.Table IYeast strainsStrainGenotypeParental strainSource/Ref.B-7528ρ+ MATa cyc1-31 cyc7-67 ura3-52 lys5-1016Moerschell R.P. Tsunasawa S. Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 524-528Crossref PubMed Scopus (132) Google ScholarB-7889ρ+ MATα cyc1-31 cyc7-67 ura3-52 leu2-3,112 can1-100B-8353ρ+ MATα CYC1 cyc7-67 ura3-52 leu2-3,112B-8354B-8354ρ+ MATα cyc1-1397 cyc7-67 ura3-52 leu2-3,112B-8514ρ+ MATa CYC1 cyc7-67 ura3-52 lys5-10B-752817Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google ScholarB-8312ρ+ MATa cyc1-1120 cyc7-67 ura3-52 lys5-10B-752817Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google ScholarB-8301ρ+ MATa cyc1-1113 cyc7-67 ura3-52 lys5-10B-752817Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google ScholarB-8634ρ+ MATa cyc1-1209 cyc7-67 ura3-52 lys5-10B-752817Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google ScholarB-8513ρ+ MATa cyc1-1195 cyc7-67 ura3-52 lys5-10B-752817Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google ScholarB-8630ρ+ MATa cyc1-1205 cyc7-67 ura3-52 lys5-10B-752817Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google ScholarB-8631ρ+ MATa cyc1-1206 cyc7-67 ura3-52 lys5-10B-752817Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google ScholarB-7734ρ+ MATa cyc1-868 cyc7-67 ura3-52 lys5-10B-7528Chen et al.aX. Chen, R. P. Moerschell, D. A. Pearce, D. D. Ramanan, and F. Sherman, unpublished data.B-7735ρ+ MATa cyc1-865 cyc7-67 ura3-52 lys5-10B-7528Chen et al.B-7686ρ+ MATa cyc1-860 cyc7-67 ura3-52 lys5-10B-7528Chen et al.B-12711ρ+ MATa cyc1-1388 cyc7-67 ura3-52 lys5-10B-7528This studyB-12712ρ- MATa cyc1-1388 cyc7-67 ura3-52 lys5-10B-7528This studyB-12713ρ+ MATa CYC1-1389 cyc7-67 ura3-52 lys5-10B-7528This studyB-12714ρ- MATa CYC1-1389 cyc7-67 ura3-52 lys5-10B-7528This studyB-12719ρ- MATa cyc1-1388 cyc7-67 ura3-52 lys5-10 sue1-1This studyB-14650ρ- MATa cyc1-1388 cyc7-67 ura3-52 sue1-1This studyB-14669ρ+ MATa his3-Δ leu2-Δ met15-Δ ura3-Δ sue1-Δ1::kanRBY4741Research GeneticsB-14705ρ+ MATa CYC1 cyc7-67 ura3-52 lys5-10 sue1-Δ1::kanRB-7528This studyB-14746ρ+ MATα cyc1-868 cyc7-67 ura3-52 leu2-3,112 can1-100B-7889This studya X. Chen, R. P. Moerschell, D. A. Pearce, D. D. Ramanan, and F. Sherman, unpublished data. Open table in a new tab Table IIcyc1 alleles Open table in a new tab Media—Escherichia coli cells containing plasmids were as grown in LB medium (10 g/liter Bacto-tryptone, 10 g/liter NaCl, and 5 g/liter yeast extract plus 100 μg/ml ampicillin or 25–50 μg/ml kanamycin) (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Standard YPD, YPS, YPG, and YPE media; synthetic minimal medium; synthetic complete medium, synthetic complete medium/Ura–, and synthetic complete medium/Leu–; and other omission media have been described by Sherman (21Sherman F. Methods Enzymol. 2002; 350: 3-41Crossref PubMed Scopus (992) Google Scholar). The media contained 1% Bacto-yeast extract, 2% Bacto-peptone, and either 2% ethanol (YPE) or 2% sucrose (YPS). Medium A was composed of synthetic minimal medium supplemented with 0.2% (w/v) casamino acids, 20 μg/ml uracil, and 2% Bactoagar. Synthetic lactate contained 0.67% Bacto-yeast nitrogen base without amino acids, 2% Bacto-agar, and 3.3% of a 30% lactate solution. Medium B was composed of synthetic lactate supplemented with 0.2% (w/v) casamino acids and 20 μg/ml uracil. Synthetic complete medium/Ura– and supplemented with 0.1% 5-fluoroorotic acid (22Boeke J.D. Lacroute F. Fink G.R. Mol. Gen. Genet. 1984; 197: 345-346Crossref PubMed Scopus (1712) Google Scholar) was also used. Construction of a Yeast Strain Containing the cyc1-1388 Allele—The desired CYC1 mutations were obtained by transforming the defective cyc1 mutant strain B-7528 (MATa cyc1-31 cyc7-67 ura3-52 lys5-10) directly with PCR-generated fragments and selection of at least partially functional cyc1 transformants, followed by sequencing the CYC1 locus of these transformants to confirm that the selected transformants contained the desired cyc1 alleles. First, the primer pair OL256 and OL257 (Table III) was used to amplify 1.7-kb fragments containing either cyc1-1209 (T78S/C102A) or CYC1-820 (C102A). Subsequently, a primer of 92 oligonucleotides was designed that contained five amino acids (RRASV) inserted between residues 4 and 5 of the CYC1 gene (primer RRASV-cyc1). Primer RRASV-cyc1 (Table III) was paired with primer OL257 to generate 653-bp fragments containing either the cyc1-1388 or CYC1-1389 allele (Table II). The B-7528 yeast strain was then transformed with either the cyc1-1388 or CYC1-1389 fragments following the procedures described by Yamamoto et al. (23Yamamoto T. Moerschell R.P. Wakem L.P. Ferguson D. Sherman F. Yeast. 1992; 8: 935-948Crossref PubMed Scopus (62) Google Scholar).Table IIIOligonucleotidesPrimerSequenceOL256GTGGACATGTCGACAATCTTACATGGOL257GCGAAGCTTGCAAATTAAAGCCTTCGRRASV-cyc1CTATAGACACGCAAACACAAATACACACACTAAATTAATAATGACTGAATTCCGTCGTGCATCTGTTAAGGCCGGTTCTGCTAAGAAAGGTGSUEF1CCCTCTGAGTGTGTGTCTGCATSUER1ACGTGGATCCCTAATGTGAGTTC151F3TCAGAGATAAGAAGAGAAAGTCOL150WFTGAGGAATCGTGCTATAAAATCAOL150WRAAGGAAACAACAAAAAGCGTT150FS1TAGTGAATAAGGATGATTTTTTAAAGAGGACAAAAATTAG150FS2CTAATTTTTGTCCTCTTTAAAAAATCATCCTTATTCACTA151ADHFTTCTTCTTCTTCGAATTCATGATTTTATTAAAGAGGACAAAAATTAGG151ADHRTTCTTCTTCTTCGAATTCTTATTTCATCTTTTTTTTGCTTTTGTGTGTC151GFPRTTCTTCTTCTTCGAATTCTTTCATCTTTTTTTTGCTTTTGTGTGTCCmycFAAGCCGTTTGACAAGGCACTGCAAATGCGGTTGACACACAAAAGCAAAAAAAAGATGAAAAGGGAACAAAAGCTGGAGCmycRATGTATGAGGAATCGTGCTATAAAATCATTTTCCATATGTATATGCCAAATATTTATTATTTACTATAGGGCGAATTGG151MYCRTTCTTCTTCTTCGAATTCTTACTATAGGGCGAATTGGGTACCG Open table in a new tab Construction of Three Sets of Isogenic Strains Containing Different cyc1 Alleles Encoding LDD, ADD, and RDD holo-1—The chromosomal DNAs were extracted from these strains containing different cyc1 alleles (see Table IV). The OL256 and OL257 primer pair (Table III) was used to amplify the 1.7-kb fragments containing the cyc1 allele of interest. Three host strains (B-7528, B-7889, and B-8354) were transformed with these 1.7-kb fragments carrying the cyc1 allele of interest. The procedures were described by Yamamoto et al. (23Yamamoto T. Moerschell R.P. Wakem L.P. Ferguson D. Sherman F. Yeast. 1992; 8: 935-948Crossref PubMed Scopus (62) Google Scholar). The corresponding ρ– strains were obtained by growing the ρ+ mutant strains in YPD plates containing ethidium bromide.Table IVConstruction of three sets of isogenic strains bearing different cyc1 alleles with or without SUE1B-7528 derivativesB-7889 derivativesB-8354 derivativesTemplate strainsNormalCYC1+CYC1+CYC1+B-8514LDDcyc1-1120 (G6I)cyc1-1120 (G6I)Not recoveredB-8312LDDcyc1-1113 (G6A)cyc1-1113 (G6A)B-8301RDDcyc1-1209 (T78S)cyc1-1209 (T78S)cyc1-1209 (T78S)B-8634RDDcyc1-1195 (T78I)cyc1-1195 (T78I)cyc1-1195 (T78I)B-8513RDDcyc1-1205 (T78G)cyc1-1205 (T78G)B-8630RDDcyc1-1206 (T78L)cyc1-1206 (T78L)B-8631ADDcyc1-868cyc1-868cyc1-868B-7734ADDcyc1-865B-7735ADDcyc1-860B-7686 Open table in a new tab Construction of the sue1-Δ1::kanR Mutant Strain by the PCR-based One-step Disruption Method—A sue1-Δ1::kanR mutant strain was purchased from Research Genetics (24Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2241) Google Scholar). The chromosomal DNA was prepared from this strain and served as a template, and the primer pair SUEF1 and SUER1 (Table III) was used to amplify the PCR-based disruption fragments carrying the sue1-Δ1::kanR mutant gene flanked by 100 bp homologous to the region directly upstream of the start codon and downstream of the stop codon of the SUE1 gene. Three sets of isogenic ρ+ cyc1 mutant strains, which were constructed as described above (see Table IV), were transformed with the amplified fragments, and G418-resistant colonies were obtained on YPD plates containing 300 μg/ml G418. Construction of the sue1-2 Mutant Strain by Site-directed Mutagenesis—The primer pair OL150WF and OL150WR (Table III) was used to produce the 1.5-kb fragments encompassing YPR150w and ∼1 kb of DNA sequence downstream of the termination codon of this gene. The 1.5-kb fragments were then inserted into the TA vector (Invitrogen). The construction was carried out with 10 ng of TA vector containing the 1.5-kb insert as a template and 125 ng of both 150FS1 and 150FS2 (Table III) as two complementary oligonucleotide primers for amplification in the presence of 2.5 units of Pfu Turbo DNA polymerase (Stratagene). The 40 nucleotides of primers 150FS1 and 150FS2 contained the desired sue1-2 mutation, a deletion of an A residue corresponding to nucleotide 9 of the YPR151c gene and to a position outside the open reading frame of YPR150w, 147 nucleotides downstream of the chain termination codon. The sue1-2 deletion of an A residue was introduced into the TA vector containing the 1.5-kb insert using a PCR-based site-directed mutagenesis kit (Stratagene) according to the instructions of the manufacturer. The TA vector with the 1.5-kb insert containing the sue1-2 allele was obtained. The 1.5-kb insert containing the sue1-2 gene was transferred to the pAB621 vector, a yeast shuttle vector containing the URA3 gene. Subsequently, the pAB621 vector with the desired insert containing the sue1-2 gene was linearized with the NheI restriction enzyme within the insert and integrated at the chromosomal SUE1 locus of the constructed yeast strain B-14746, an isogenic strain of B-7889 containing the cyc1-868 allele. The sequence of the sue1-2 allele was verified by examining the appropriate PCR product. Construction of a Series of Subclones in the YCp50 Vector—Plasmids pAB2930, pAB2931, and pAB2933 (see Fig. 1) were constructed by digestion of pAB2927 with SphI, NruI, and EcoRI, respectively, followed by gel purification of large fragments of ∼11, 13, and 11 kb, respectively, and subsequent recircularization with T4 DNA ligase (New England Biolabs Inc.). Plasmids pAB2930, pAB2931, and pAB2933 are YCp50-based centromere plasmids containing the URA3 gene. For plasmids pAB2932 and pAB2934, ∼4.5-kb BamHI and 2.7-kb HindIII fragments of pAB2927, respectively, were gel-purified and ligated into their corresponding sites in the polylinker of the YCp50 vector (see Fig. 1). Construction of the C-terminal Green Fluorescent Protein (GFP)-fused SUE1 Gene—Approximately 640-bp fragments containing the SUE1 gene (YPR151c without a stop codon) flanked with EcoRI restriction sites were amplified using the primer pair 151ADHF and 151GFPR (Table III) and subsequently digested with the EcoRI restriction enzyme. The gel-purified EcoRI fragments were then ligated to the EcoRI site in the polylinker of plasmid pGFP-C-FUS (pAA1931), giving rise to the C-terminal GFP-fused SUE1 gene under the control of the Pmet25 promoter. Plasmid pAB1931 carrying C-terminal GFP-SUE1 (pAB2935) was obtained from the ampicillin-resistant colonies. Both plasmids pAB1931 and pAB2935 were transformed into strain B-14705, an isogenic strain of B-7528 containing the CYC1 and sue1-Δ1::kanR genes. Colonies containing plasmid pAB1931 or pAB2935 were selected on synthetic complete medium/Ura– plates. Construction of the C-terminal Myc-tagged SUE1 Gene—Initially, the yeast strain bearing the C-terminal (Myc)3-tagged SUE1 gene was constructed. A (Myc)3-tagging cassette in plasmid pMPY-3×Myc (pAA1869) (25Schneider B.L. Seufert W. Steiner B Yang Q.H. Futcher A.B. Yeast. 1995; 11: 1265-1274Crossref PubMed Scopus (291) Google Scholar) was used as a template, and the following primer pair was designed: CmycF, with the 5′ 60 bases homologous to sequences immediately prior to the stop codon of SUE1 and the 3′ 18 bases complementary to unique sequences in the (Myc)3-tagging cassette; and CmycR, with the 5′ 63 bases homologous to sequences immediately 3′ of the stop codon of SUE1 and the 3′ 16 bases complementary to unique sequences in the (Myc)3-tagging cassette. PCR conditions were as described by Schneider et al. (25Schneider B.L. Seufert W. Steiner B Yang Q.H. Futcher A.B. Yeast. 1995; 11: 1265-1274Crossref PubMed Scopus (291) Google Scholar). The gel-purified 1.5-kb fragments were transformed into strain B-14803. Ura+ colonies were selected and then grown overnight in YPD medium, and cultures were washed with water and spread onto 5-fluoroorotic acid plates. The 5-fluoroorotic acid-resistant colonies were selected and subsequently confirmed by sequencing to contain the integrated C-terminal (Myc)3-tagged SUE1 gene without the URA3 gene. We did not detect any signal of Myc-tagged SUE1 in whole cell extracts using monoclonal antiserum (purchased from NeoMarkers) against the Myc tag. Thus, we tried to construct C-terminal (Myc)3-tagged SUE1 in the 2μ-based yeast expression vector pBEVY containing the URA3 gene (pAB2324) (26Miller III, C.A. Martinat M.A. Hyman L.E. Nucleic Acids Res. 1998; 26: 3577-3583Crossref PubMed Scopus (104) Google Scholar), and we also constructed SUE1 in the same vector (pAB2324) as a control. The total DNA prepared from the strain containing (Myc)3-tagged SUE1 was used as a template, and the primer pair 151ADHF and 151MYCR (Table III) was designed. Approximately 820-bp fragments containing C-terminal (Myc)3-tagged SUE1 flanked by EcoRI restriction sites were amplified, subsequently digested with the EcoRI restriction enzyme, and ligated to the EcoRI site in the polylinker of the pAB2324 vector. Meanwhile, wild-type SUE1 was also amplified using the primer pair 151ADHF and 151ADHR (Table III). Approximately 650-bp fragments containing SUE1 flanked by EcoRI restriction sites were amplified, digested, and ligated to the EcoRI site in the polylinker of pAB2324 as well. The plasmids containing either (Myc)3-tagged SUE1 (pAB2936) or SUE1 (pAB2937) under the control of the ADH1 (alcohol dehydrogenase-1) promoter were obtained from the ampicillin-resistant colonies and transformed into strain B-14705, an isogenic strain of B-7528 containing the CYC1 and sue1-Δ1::kanR genes. Strain B-14705 with pAB2936 or pAB2937 was selected on plates of synthetic dropout medium/Ura–. Determination of holo-1 Content—holo-1 levels in intact cells were screened by the benzidine staining method (17Pearce D.A. Sherman F. Arch. Biochem. Biophys. 1998; 352: 85-96Crossref PubMed Scopus (4) Google Scholar) and examined visually with a spectroscope as described by Sherman and Slonimski (27Sherman F. Slonimski P.P. Biochim. Biophys. Acta. 1964; 90: 1-15Crossref PubMed Scopus (165) Google Scholar). More accurate estimation was performed by the method of low temperature (–196 °C) spectrophotometric recording with a modified Aviv Model 14DS spectrophotometer as described by Hickey et al. (28Hickey D.R. Jayaraman K. Goodhue C.T. Shah J. Clements J.M. Tsunasawa S. Sherman F. Gene (Amst.). 1991; 105: 73-81Crossref PubMed Scopus (40) Google Scholar). Western Blotting and Immunological Procedures—Samples were electrophoresed on an SDS-4–20% gradient acrylamide gel, transferred to a Hybond-ECL membrane (Amersham Biosciences), and probed with antiserum, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad). Anti-cytochrome c polyclonal antibodies were diluted 1:4000 in 0.1% Tween 20 in Tris-buffered saline (TBS; 20 mm Tris (pH 7.6) and 137 mm NaCl); anti-Myc monoclonal antibodies were diluted 1:100 in 0.1% Tween 20 in TBS; and anti-His monoclonal antibodies were diluted 1:2000 in 0.1% Tween 20 in TBS. A Hybond-ECL membrane was incubated with diluted antibodies for 1 h in 0.1% Tween 20 in TBS. The secondary antibodies were used at 1:2000 for 1 h. The membrane was washed with 0.1% Tween 20 in TBS, followed by ECL detection (Amersham Biosciences). Preparation of Yeast Mitochondria and the IMS of Mitochondria— Yeast strain B-14705 with plasmid pAB2936 or pAB2937 containing the SUE1 constructs was expressed at high levels under the control of the constitutive ADH1 promoter. Mitochondrial purification and subfractionation were carried out following the method described by Zinser and Daum (29Zinser E. Daum G. Yeast. 1995; 11: 493-536Crossref PubMed Scopus (305) Google Scholar). Detection of GFP-fused Sue1p in Yeast Cells—Yeast strain B-14705 containing either pAB1931 or pAB2935 was grown overnight on synthetic complete medium/Ura– plates and diluted with distilled water to the appropriate concentration, followed by continuous growth in synthetic complete medium/Ura– overnight to A600 = 0.1–0.2. Cells were harvested and suspended at a concentration of 106 cells/ml in 10 mm HEPES (pH 7.4) and 5% glucose. Rhodamine B (hexyl ester; Molecular Probes, Inc.) was added to a final concentration of 100 nm. The mixture was incubated at room temperature for 20 min. Yeast cells were harvested by centrifugation at 13,000 rpm for 5 s; washed; suspended in a small volume of 10 mm HEPES (pH 7.4) and 5% glucose; and visualized by confocal microscopy on a Leica TCS SP microscope equipped with argon, krypton/argon, and ultrav
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