The yeast Saccharomyces cerevisiaecontains two forms of cytochrome c, iso-1- and iso-2-cytochrome c, which are encoded by the nuclear genesCYC1 and CYC7, respectively. The cytochromesc are synthesized in the cytosol, imported into mitochondria, and subsequently modified by the covalent attachment of heme through the action of cytochrome c heme lyase, which is encoded by CYC3. Apo-iso-2-cytochrome c but not apo-iso-1-cytochrome c was observed incyc3 − mutants. Furthermore, pulse-chase experiments previously demonstrated that the lack of apo-iso-1-cytochrome c was due to its rapid degradation. We report herein that this degradation of apo-iso-1-cytochromec is dependent on ubiquitination and on the action of the proteasome. Diminished degradation of apo-iso-1-cytochromec was observed in pre2-2 and pre1-1mutants having altered proteasome subunits; in ubc1,ubc4, and ubc5 strains lacking one or more of the ubiquitin-conjugating enzymes; and in strains blocked in multi-ubiquitination by overproduction of the abnormal ubiquitin-K48R ubiquitin. In addition, we have used epitope-tagged ubiquitin to demonstrate that apo-iso-1-cytochrome c but not apo-iso-2-cytochrome c is ubiquitinated. Furthermore, the degradation of apo-iso-1-cytochrome c was diminished when the N-terminal region was replaced with the N-terminal region of apo-iso-2-cytochrome c, indicating that this region may be the target for degradation. We suggest that ubiquitin-dependent degradation of apo-iso-1-cytochromec is part of the regulatory process controlling the preferential expression of the iso-cytochromes c. The yeast Saccharomyces cerevisiaecontains two forms of cytochrome c, iso-1- and iso-2-cytochrome c, which are encoded by the nuclear genesCYC1 and CYC7, respectively. The cytochromesc are synthesized in the cytosol, imported into mitochondria, and subsequently modified by the covalent attachment of heme through the action of cytochrome c heme lyase, which is encoded by CYC3. Apo-iso-2-cytochrome c but not apo-iso-1-cytochrome c was observed incyc3 − mutants. Furthermore, pulse-chase experiments previously demonstrated that the lack of apo-iso-1-cytochrome c was due to its rapid degradation. We report herein that this degradation of apo-iso-1-cytochromec is dependent on ubiquitination and on the action of the proteasome. Diminished degradation of apo-iso-1-cytochromec was observed in pre2-2 and pre1-1mutants having altered proteasome subunits; in ubc1,ubc4, and ubc5 strains lacking one or more of the ubiquitin-conjugating enzymes; and in strains blocked in multi-ubiquitination by overproduction of the abnormal ubiquitin-K48R ubiquitin. In addition, we have used epitope-tagged ubiquitin to demonstrate that apo-iso-1-cytochrome c but not apo-iso-2-cytochrome c is ubiquitinated. Furthermore, the degradation of apo-iso-1-cytochrome c was diminished when the N-terminal region was replaced with the N-terminal region of apo-iso-2-cytochrome c, indicating that this region may be the target for degradation. We suggest that ubiquitin-dependent degradation of apo-iso-1-cytochromec is part of the regulatory process controlling the preferential expression of the iso-cytochromes c. The yeast Saccharomyces cerevisiae contains two forms of cytochrome c, iso-1-cytochrome c and iso-2-cytochrome c, which are encoded by the nuclear genesCYC1 and CYC7, respectively. Iso-1-cytochromec and iso-2-cytochrome c, which are 80% identical, normally comprise 95 and 5% of total cytochromec, respectively, in aerobically grown, derepressed cells (1Sherman F. Taber H. Campbell W. J. Mol. Biol. 1965; 13: 21-39Crossref PubMed Scopus (75) Google Scholar). Both iso-cytochromes c are synthesized in the cytosol, as apo-cytochrome c, and subsequently translocated into the mitochondria. Heme is covalently attached to the apo-cytochromesc by cytochrome c heme lyase (CCHL), 1The abbreviations used are: CCHL, cytochromec heme lyase; apo-1, apo-iso-1-cytochrome c; apo-2, apo-iso-2-cytochrome c; HA, the hemagglutinin epitope YPYDVPDYA; Ub, normal synthetic ubiquitin; Ub-K48R, Ub with a K48R replacement; HA-Ub, Ub with the HA attached to the N terminus of Ub; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; E2, ubiquitin-conjugating enzymes; E3, ubiquitin-protein ligase. which is encoded by the gene CYC3, resulting in the formation of the mature holo-cytochromes c (2Dumont M.E. Ernst J.F. Hampsey D.M. Sherman F. EMBO J. 1987; 6: 235-241Crossref PubMed Scopus (165) Google Scholar). Import of the apo-cytochromesc is dependent on the action of CCHL, andcyc3-Δ mutants, lacking CCHL, accumulate apo-cytochromesc in the cytosol (3Dumont M.E. Ernst J.F. Sherman F. J. Biol. Chem. 1988; 263: 15928-15937Abstract Full Text PDF PubMed Google Scholar). However, apo-1 (apo-iso-1-cytochromec) is not detected in cyc3-Δ mutants, whereas apo-2 (apo-iso-2-cytochrome c) is present at the corresponding level of holo-iso-2-cytochrome c in relatedCYC3 + strains. Dumont et al. (4Dumont M.E. Matthews 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-1 is rapidly degraded. In this study, we have demonstrated that apo-1 degradation requires functional proteosomes and is mediated by the ubiquitin-dependent pathway. The ubiquitin system is the major pathway for targeting and selective degradation of proteins in the cytosol and nucleus of eukaryotes (5Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1600) Google Scholar, 6Finley D. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces cerevisiae, Gene Expression. 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 539-581Google Scholar, 7Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1205) Google Scholar, 8Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-223Crossref PubMed Scopus (784) Google Scholar, 9Hochstrasser M. Cell. 1996; 84: 813-815Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 10Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1459) Google Scholar, 11Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar). Ubiquitin is a 76-residue protein that is joined reversibly to protein substrates by linkage between the α-carboxyl group of ubiquitin and ε-amino groups of certain lysine residues of the acceptor proteins through an isopeptide bond in a multistep reaction. Ubiquitin is first activated through the formation of of a thiol ester bond of C-terminal glycine by a ubiquitin-activating enzyme. Ubiquitin then forms a thiol ester bond with a second protein, one of the ubiquitin-conjugating enzymes, E2. The transfer of ubiquitin to protein substrates by an E2 is done with or without the participation of a ubiquitin-protein ligase, E3. Additional ubiquitin molecules are usually added to the substrate by the same enzyme cascade, in which ubiquitin moieties are linked to the lysine 48 of each previously conjugated ubiquitin molecule until multi-ubiquitin chains are formed, which facilitates a more rapid degradation by the action of the 26 S proteosome or sometimes by the action of vacuoles. The conclusions on the degradation of apo-1 and the lack of degradation of apo-2 were based on the use of defined conditional mutants of the proteosome, on mutants lacking one of more of the E2-conjugating enzymes, on the inhibition of multi-ubiquitination by overexpression of the altered ubiquitin Ub-K48R, and on the identification of ubiquitinated forms of apo-1 but not apo-2. Because multi-ubiquitinated proteins also can be degraded by the action of vacuoles (or lysosomes), we have also examined pep4-Δ mutants, which are defective in a subset of vacuolar proteinases and which are reduced in the degradation of numerous proteins. The yeast strains used in this study are listed in Table I. Strain WCG4-11/22a, obtained from Dr. Dieter H. Wolf, along with our laboratory strains, were used to construct pre2-2 cyc7-Δand pre1-1 cyc7-Δ strains. CYC3 +genes were disrupted by transforming ura3-52 strains with a 3-kilobase pair EcoRI-XhoI linear fragment, which was derived from the plasmid pAB609 and which corresponds to a segment encompassing the CYC3 gene in which the coding region was replaced by the URA3 gene. Strains B-10105 through B-10108, B-10109 through B-10112, and B-101155 through B-10158 represent three tetrads resulting from a cross of B-7553 and B-10104. Strains B-10120 through B-10124 were prepared by disrupting one or more of theUBC1, UBC4, and UBC5 genes in strain B-8107 with, respectively, the following plasmids treated with the following endonucleases: pS-UBC1, HindIII; pUBC4HIS3,SphI-EcoRI; and pUBCLEU2,SacI-PstI (see Refs. 12Seufert W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (407) Google Scholar and 13Seufert W. McGrath J.P. Jentsch S. EMBO J. 1990; 9: 4535-4541Crossref PubMed Scopus (122) Google Scholar). YEp96 (also denoted p[PCUP1-Ub]), YEp110 (also denoted p[PCUP1-Ub-K48R), and YEp112 (also denoted p[PCUP1-HA-Ub]) are 2-μm plasmids that encode normal (Ub), Ub-K48R, and HA-Ub, respectively, under the control of the copper-inducible CUP1 promoter, as described by Hochstrasseret al. (14Hochstrasser M. Ellison M.J. Chau V. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4021-4610Crossref Scopus (197) Google Scholar). Ub denotes normal ubiquitin; Ub-K48R denotes Ub with a K48R replacement; and HA-Ub denotes ubiquitin with a YPYDVPDYA epitope from hemagglutinin (HA) of influenza virus attached to the N terminus of Ub. Disruptions and plasmids were introduced by conventional transformation procedures.Table IYeast strains used in this studyStrain no.GenotypeWCG4–11/22aMAT a a a a a a a or a or a or a or a a a a a plasmid YEp96 (also denoted CUP1 plasmid YEp110 (also denoted CUP1 plasmid YEp112 (also denoted CUP1 plasmid YEp96 (also denoted CUP1 plasmid YEp110 (also denoted CUP1 plasmid YEp112 (also denoted CUP1 plasmid YEp96 (also denoted CUP1 a a a a + a + a a a a a a plasmid YEp96 (also denoted CUP1 a a a a a in a The were by transforming strain a ura3-52 S. Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1988; PubMed Scopus Google Scholar) with synthetic as described by al. D. Sherman F. 1992; PubMed Scopus Google Scholar). the CYC3 gene was disrupted in each of the strains as described resulting in strains to Strain is to this The or were or from the of the fragment, as with of the obtained with the as described previously Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). The pre2-2 and pre1-1 were by the encompassing and respectively, the chain F. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) with a S. the of was by of the and of apo-1 from strains pre2-2 or strains were with yeast to in of and and of apo-1 from strains p[PCUP1-Ub-K48R), and plasmids were with yeast to in of yeast and the were by at for in and in the same of the and the was for an yeast strains were at for pre2-2 strains and control strains, which were at c was prepared as described by and Sherman Sherman F. J. Biol. Chem. Full Text PDF Google Scholar). to the HA epitope were obtained from Yeast strains as described were by in of PubMed Scopus Google lacking yeast and of was added to a of and the cells were for an by a in of cells were at the by at for in and in of were in an of the on for of was added to the and protein on each was at the was and the was in of and at for The was more in to and subsequently by for in and The were at for and the was as described previously for c Sherman F. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). were on a gel H. Biochem. 1987; Scopus Google the were apo-1 was by and the of the was by Yeast were in the and proteins were or and on a as described were to by J. Molecular Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar). protein was blocked by of in or 5% The were for with or c at of or respectively. The were or or of the level and of described apo-1 is rapidly degraded in the when into the and is blocked by the which In apo-2 is not degraded under (4Dumont M.E. Matthews 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). We have the pathway of degradation by or not the level of apo-1 is diminished in strains having or in the following iso-1-cytochrome c having the defective encoded by pre2-2 and due to the pep4-Δ the ubiquitin Ub-K48R, which and the lack of one or more of the E2-conjugating and were with strains lacking due to and lacking apo-2 due to the or cyc7-Δ The of apo-1 in + cyc3-Δ cyc7-Δ were by and the of apo-1 was by pulse-chase we apo-1 is for degradation through the ubiquitin-dependent apo-2 is by apo-1 with altered N-terminal the N-terminal region corresponding to apo-2. that the in iso-1-cytochrome c as a at at three in the molecule Hampsey D.M. Biochem. 1991; PubMed Scopus Google Scholar, D. Sherman F. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and are three that in is at by the in Furthermore, the iso-1-cytochrome c an in Sherman F. Mol. Genet. 1995; PubMed Scopus Google Scholar). In addition, the an in the of the D. Sherman F. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). of of that are the in the iso-1-cytochrome c D. Sherman F. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, Sherman F. 1995; PubMed Scopus Google Scholar). We have examined apo-1 to the degradation may be dependent on the of the The of the that the normal apo-1 and the apo-1 in strains B-8107 and respectively, were degraded not indicating that the degradation may not be dependent on the the in apo-1 and apo-2 degradation be by the in However, we have not demonstrated the apo-cytochrome c, as for c. We have that functional are for degradation of apo-1 by the of apo-1 and pre1-1 mutants having defective W. J. EMBO J. 1991; PubMed Scopus Google Scholar, W. A. V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The pre2-2 corresponds to a at an in the whereas the pre1-1 corresponds to a at a in the W. J. EMBO J. 1991; PubMed Scopus Google Scholar, W. A. V. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of apo-1 were in pre2-2 + cyc7-Δ cyc3-Δ + + cyc7-Δ cyc3-Δ strains that were derived as from a cross the following two + cyc7-Δ and B-7553 + + cyc7-Δ The were for the B-10105 through B-10108, B-10109 through B-10112, and through corresponding to three The were from the and were by the encompassing in of apo-1 in + cyc7-Δ cyc3-Δ in the of apo-1 in the pre2-2 strains and as + strains also were obtained and pre1-1 pre2-2 + cyc7-Δ cyc3-Δ not Furthermore, pulse-chase that rapid degradation of apo-1 + strains and with pre2-2 and that the proteasome is in of ubiquitin-conjugating are in ubiquitination of of strains, to the control strain B-8107 + cyc7-Δ were prepared by disrupting one or more of the genes UBC4, as in the with the following of genes encode an E2 ubiquitin-conjugating enzyme. The strains were to at in The cells were in PubMed Scopus Google Scholar) and and with and in at the were with and by in a apo-1 was by of apo-1 degradation in the following c, and pre2-2 the degradation of are for the following + cyc3-Δ cyc7-Δ derived from a + and B-10109 were to in at and in PubMed Scopus Google Scholar) and and subsequently with and in at the were with c and by in a and and respectively, of apo-1 degradation in the following strains described and We have that the level of apo-1 was not in pep4-Δ strains, which are in a subset of vacuolar proteinases that numerous proteins to the certain that are ubiquitinated Mol. Cell. Biol. 6: PubMed Scopus Google Scholar, S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell Biol. PubMed Scopus Google Scholar, Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar, H. Cell. 1996; 84: Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, D. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). of apo-1 were in pep4-Δ + cyc7-Δ cyc3-Δ + + cyc7-Δ cyc3-Δ strains that were derived as from a cross the following two B-8107 + + cyc7-Δ and + + The were for and of apo-1 were to be in pep4-Δ + cyc7-Δ cyc3-Δ and strains + + cyc7-Δ cyc3-Δ and strains. was disrupted in strain B-8107 by of The level of apo-1 in was to the apo-1 level in the + strain the of apo-1 in pep4-Δ + strains that the is not in the degradation of We have that ubiquitination is for degradation of apo-1 by the of apo-1 in a of strains lacking one or more of the E2-conjugating and which are for the of proteins and which W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (407) Google W. McGrath J.P. Jentsch S. EMBO J. 1990; 9: 4535-4541Crossref PubMed Scopus (122) Google Scholar). The and UBC5 + genes were disrupted in strain B-8107 + cyc7-Δ or in experiments with cells that were the of in that the degradation of apo-1 was diminished to a in each of and mutants and that this was more in the and and mutants. In addition, multi-ubiquitination was by Ub-K48R, an altered ubiquitin in which the normal lysine 48 was replaced by Ub-K48R ubiquitin can to proteins but is to as an acceptor for multi-ubiquitination V. A. D. Varshavsky A. PubMed Scopus Google J. PubMed Google Scholar). B-8107 + cyc7-Δ was with the plasmids and which have the normal Ub and Ub-K48R respectively, under control of the promoter, as described by et al. (14Hochstrasser M. Ellison M.J. Chau V. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4021-4610Crossref Scopus (197) Google Scholar). in demonstrated that degradation is diminished in the of The in apo-1 degradation in and the in the and the that ubiquitin was in experiments described in with the mutants and the Ub-K48R ubiquitin that ubiquitination is in the degradation of of proteins detected by ubiquitin or epitope-tagged ubiquitin (14Hochstrasser M. Ellison M.J. Chau V. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4021-4610Crossref Scopus (197) Google Scholar, J. J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). We have demonstrated that apo-1 is ubiquitinated by normal ubiquitin and a HA epitope-tagged ubiquitin under control of the promoter, as described by et al. (14Hochstrasser M. Ellison M.J. Chau V. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4021-4610Crossref Scopus (197) Google Scholar). The strains + cyc7-Δ cyc3-Δ and cyc7-Δ lacking and apo-1, respectively, and and B-8107 + cyc7-Δ also lacking and apo-1, respectively, were with the CUP1 plasmid normal ubiquitin. in protein with an that apo-1 is present in the and strains + cyc7-Δ and respectively, but not in the and strains and respectively. The in represent ubiquitin of of which contains the pre2-2 a that may represent the and of of is the of the rapid degradation which is diminished in and which a was not observed in + strain control not to this that a of ubiquitin was a apo-1 with that under degradation is In addition, a control strain apo-2 and not apo-1 a of apo-2 when or of ubiquitin were was of the lack of degradation of apo-2 as described previously (4Dumont M.E. Matthews 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). apo-2 is not a substrate for ubiquitination but as a control for In a the strains and B-8107 + cyc7-Δ were with the CUP1 plasmid ubiquitin with an HA The proteins were with c by and were with or as in The of apo-1 was on one of the gel by with c, whereas the of the ubiquitinated apo-1 was on the by with are in and and and and The ubiquitinated of apo-1 is with proteins from strain + cyc7-Δ cyc3-Δ p[PCUP1-HA-Ub]) but not with proteins from the control strains and The of the ubiquitinated apo-1 was the of apo-1, that apo-1 was However, of the and of ubiquitin by is to be J. Biochem. 1995; PubMed Scopus Google Scholar). were observed with experiments on numerous with of the and with that of apo-1 not in and demonstrated that apo-1 is ubiquitinated. In to the of apo-1 that was a of multi-ubiquitinated forms was in was used to the of the following that the degradation of apo-1 was diminished when the the N-terminal region was replaced with the N-terminal region of apo-2 apo-1 was at the same level + apo-1 in pre2-2 Furthermore, the and c, but not the apo-1, also that the normal the N-terminal region of apo-1 is at in part the target for ubiquitin-dependent degradation. We have that of apo-1 requires functional and that this degradation is mediated by the ubiquitin pathway. Diminished degradation of apo-1 in pre1-1 strains having altered proteasome subunits; and ubc5 strains lacking one or more of the ubiquitin enzymes; and in strains blocked in ubiquitination by overproduction of the abnormal Ub-K48R ubiquitin. In addition, overproduction of normal Ub and epitope-tagged HA-Ub ubiquitin was used to demonstrate that apo-1 is ubiquitinated. Furthermore, the inhibition of degradation by Ub-K48R ubiquitin and the of of apo-1, that multi-ubiquitination may be the Because the pep4-Δ on apo-1 the not to a in the degradation. the not apo-1 from degradation. In the certain forms of c from degradation that mitochondria. A. and F. The to rapidly certain proteins to be in S. Rev. 1992; PubMed Google Scholar). of the protein degradation by ubiquitination and subsequently by the proteosome have that regulatory as as abnormal are for degradation W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (407) Google Scholar, M. Ellison M.J. Chau V. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4021-4610Crossref Scopus (197) Google Scholar, A. D. Varshavsky A. Cell. Full Text PDF PubMed Scopus Google Scholar, M. Varshavsky A. Cell. 1990; 61: Full Text PDF Scopus Google Scholar). study on apo-1 the first of a protein that is degraded by the ubiquitin-dependent pathway. Furthermore, we suggest that the degradation of apo-1 and apo-2 is part of the regulatory process controlling the preferential expression of the iso-cytochromes c. or the of iso-1-cytochrome c but the of iso-2-cytochrome c and the of iso-2-cytochrome c can be F. Annu. Rev. Genet. PubMed Scopus Google Scholar). is by the major for the expression of the iso-cytochromes c, we suggest that the degradation of apo-1 and apo-2 also may a and the of heme and are to the of apo-cytochrome c. Because iso-2-cytochrome c is under is to the of the cells to apo-1, resulting in a of apo-2 in a of iso-2-cytochrome c. of preferential degradation of apo-1 is with of iso-2-cytochrome c in mutants which are in mutants and certain mutants having of (1Sherman F. Taber H. Campbell W. J. Mol. Biol. 1965; 13: 21-39Crossref PubMed Scopus (75) Google Scholar, M.E. Matthews 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, M.E. Sherman F. Mol. Cell. Biol. 13: PubMed Scopus Google Scholar). major the identification of the for degradation that is present in apo-1 but in In the of protein substrates for ubiquitin-dependent degradation is and as a of proteins for degradation have by the ubiquitin system requires at two a for a E2-conjugating or a protein and an acceptor encompassing a lysine for The two can be in or and can in of an protein Varshavsky A. 1990; Scholar). The of the may be due in part to the numerous E2, which are encoded by a at in yeast M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1459) Google Scholar). of degradation have in with that proteins S. D. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar, A. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). degradation the A. Cell. 1992; Full Text PDF PubMed Scopus Google also denoted S. D. Mol. Cell. Biol. 1995; PubMed Scopus Google in which and in the degradation of based on the of at the N of also in a of and and or S. D. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar). are linear of as and and and for degradation of proteins have and by the and of M. H. EMBO J. PubMed Scopus Google a the S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar, M. 1991; PubMed Scopus Google Scholar, EMBO J. 1991; PubMed Scopus Google Scholar, H. D. EMBO J. 1994; 13: PubMed Scopus Google residues and of the protein M. D. Cell. 1994; Full Text PDF PubMed Scopus Google and which contains two degradation one between and and a second between and M. Varshavsky A. Cell. 1990; 61: Full Text PDF Scopus Google Scholar). In addition, et al. S. M. PubMed Scopus Google Scholar) that proteins a region in residues of and of the apo-1 and apo-2 which are 80% identical, not that can the ubiquitin-dependent degradation. c are apo-1 and apo-2, which and lysine respectively. contains lysine residues at and which are in apo-2, whereas apo-2 contains lysine residues at and which are in major between the iso-cytochromes c is that apo-2 contains residues at the N We have examined the of the N-terminal region by the of apo-1 of altered forms in in The in the three of the altered and c, but not the apo-1, that N-terminal region of apo-1 is at in part in the ubiquitin-dependent degradation. apo-1, an altered N-terminal region corresponding to the normal apo-2 N-terminal the in degradation be that the level of apo-1 is to the level of apo-2 in the that holo-iso-2-cytochrome c 5% of the c and that the level of apo-2 in cyc3-Δ strains is to the level of holo-iso-2-cytochrome c + strains. the N-terminal region of apo-1 with the N-terminal region of apo-2 in a degradation. The for the ubiquitin-dependent in to the N-terminal to be We Dr. Dieter H. for yeast strain the pre1-1 and Dr. Jentsch for plasmids pS-UBC1, and used in the of the and Dr. of for plasmids and YEp112 respectively, normal (Ub), Ub-K48R, and HA-Ub ubiquitin. were by S. of of and and Dr. and the CYC3 in strains to were by Dr. M. Dumont of of and
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