Ubiquitin conjugation to cytochromes c. Structure of the yeast iso-1 conjugate and possible recognition determinants.

Saccharomyces cerevisiae iso-1-cytochrome c was conjugated with ubiquitin (Ub) in vitro in a rabbit reticulocyte extract (Fraction II). By N-terminal protein sequencing, it was found for both the mono- and diubiquitinated products that the major Ub attachment site is on Lys4 (residue 9) of the cytochrome c. Thus, the residue ubiquitinated in iso-1-cytochrome c is identical with that previously determined for the yeast iso-2 form (Sokolik, C. W., and Cohen, R. E. (1991) J. Biol. Chem. 266, 9100-9107). For both cytochromes c, the proportions of diubiquitinated and higher order conjugates are drastically reduced when Ub is replaced with a Lys48----Arg variant, suggesting that the Ub-Ub moieties are linked predominantly through Lys48. Despite close similarities in structure and ubiquitination sites, conjugation to iso-2-cytochrome c is approximately 5-fold faster than for the iso-1 form; vertebrate cytochromes c are even poorer substrates, being ubiquitinated at only approximately 5% of the rate of the iso-2 protein. Comparison of several cytochrome c variants excludes alpha-N-acetylation or the identity of the N-terminal amino acid as the important recognition determinants in these reactions. The results, which include the finding that ferro and ferri-iso-2-cytochromes c are ubiquitinated equally, also are evidence against a simple correlation between ubiquitination efficiency and thermodynamic stability. Rather, the presence of a pair of lysines (Lys4-Lys5) within the relatively unstructured N-terminal extension of the yeast cytochromes c may be responsible for their preferential ubiquitination.     

Replacement of the invariant lysine 77 by arginine in yeast iso-1-cytochrome c results in enhanced and normal activities in vitro and in vivo

Oligonucleotide-directed mutagenesis of the yeast Saccharomyces cerevisiae was used to generate an abnormal iso-1-cytochrome c having an Arg-77 replacement of the normal Lys-77; this Lys-77 residue is evolutionarily conserved in most eukaryotic cytochromes c and is trimethylated in fungal and plant cytochromes c. Examination of strains having a single chromosomal copy of the gene encoding the Arg-77 protein indicated that the altered protein was synthesized at the normal rate and that it had normal or near normal activity in vivo. Examination of enzymatic activities in vitro with cytochrome b2, cytochrome c peroxidase, and cytochrome c oxidase indicated that the altered iso-1-cytochrome c has equal or enhanced catalytic efficiencies. Thus, replacement of the evolutionarily conserved residue Lys-77 produces no or only minor effects both in vivo and in vitro.     

Cytochrome c methyltransferase, Ctm1p, of yeast

Cytochromes c from plants and fungi, but not higher animals, contain methylated lysine residues at specific positions, including for example, the trimethylated lysine at position 72 in iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. Testing of 6,144 strains of S. cerevisiae, each overproducing a different open reading frame fused to glutathione S-transferase, previously revealed that YHR109w was associated with an activity that methylated horse cytochrome c. We show here that this open reading frame, denoted Ctm1p, is specifically responsible for trimethylating lysine 72 of iso-1-cytochrome c. Unmethylated forms of cytochrome c but not other proteins or nucleic acids are methylated in vitro by Ctm1p produced in S. cerevisiae or Escherichia coli. Iso-1-cytochrome c purified from a ctm1-Delta strain is not trimethylated in vivo, whereas the K72R mutant form, or the trimethylated Lys-72 form of iso-1-cytochrome c, are not significantly methylated by Ctm1p in vitro. Like apocytochrome c, but in contrast to holocytochrome c, Ctm lp is located in the cytosol, consistent with the view that the natural substrate is apocytochrome c. The ctm1-Delta strain lacking the methyltransferase did not exhibit any growth defect on a variety of media and growth conditions, and the unmethylated iso-1-cytochrome c was produced at the normal level and exhibited the normal activity in vivo. Ctm1p and cytochrome c were coordinately regulated during anaerobic to aerobic transition, a finding consistent with the view that this methyltransferase evolved to act on cytochrome c.     

Genes Affecting the Expression of Cytochrome c in Yeast: Genetic Mapping and Genetic Interactions

The four mutant genes, cyc2, cyc3, cyc8 and cyc9, that affect the levels of the two iso-cytochromes c in the yeast Saccharomyces cerevisiae have been characterized and mapped. Both cyc2 and cyc3 lower the amount of iso-1-cytochrome c and iso-2-cytochrome c; whereas, cyc8 and cyc9 increase the amount of iso-2-cytochrome c. The cyc2, cyc3, cyc8 and cyc9 genes are located, respectively, on chromosomes XV, I, II and III, and are, therefore, unlinked to each other and unlinked to CYC1, the structural gene of iso-1-cytochrome c and to CYC7, the structural gene of iso-2-cytochrome c. While some cyc3 mutants are completely or almost completely deficient in cyotchromes c, none of the cyc2 mutants contained less than 10% of parental level of cytochrome c even though over one-half of the mutants contain UAA or UAG nonsense mutations. Thus, it appears as if a complete block of the cyc2 gene product still allows the formation of a residual fraction of cytochrome c. The cyc2 and cyc3 mutant genes cause deficiencies even in the presence of CYC7, cyc8 and cyc9, which normally cause overproduction of iso-2-cytochrome c. We suggest that cyc2 and cyc3 may be involved with the regulation or maturation of the iso-cytochromes c. In addition to having high levels of iso-2-cytochromes c, the cyc8 and cyc9 mutants are associated with flocculent cells and other abnormal phenotypes. The cyc9 mutant was shown to be allelic with the tup1 mutant and to share its properties, which include the ability to utilize exogenous dTMP, a characteristic flocculent morphology, the lack of sporulation of homozygous diploids and low frequency of mating and abnormally shaped cells of alpha strains. The diverse abnormalities suggest that cyc8 and cyc9 are not simple regulatory mutants controlling iso-2-cytochrome c.     

Network of interactions between unlinked genes: synergistic and antagonistic regulation of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 synthesis]

Five chromosomal genes, CYPI to CYP5 involved in the regulation of the synthesis of iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2 are described. The function of these genes was studied either by varying the proportion of the mutated and wild type alleles in the cell vy varing the growth conditions, or else by transforming the mutants into sigma-cytoplasmic petites. We have shown a network of genetic interactions which regulate the synthesis of three structurally different proteins : iso-1-cytochrome c, iso-2-cytochrome c and cytochrome b2, by two unlinked genes : CYC1 and CYP1, one of which (CYC1) is the structural gene by iso-1-cytochrome c. Within this network the interactions are proportional to the gene dosage and are either antagonistic or synergistic depending on the allele combination and the protein studied. The mutated alleles cyp1 stimulate the synthesis of iso-2-cytochrome c, inhibit the synthesis of iso-1-cytochrome c, while the cytochrome b2 synthesis is also inhibited but by a combination of cyp1 mutated alleles CYC1 wild type allele. Other loci, CYP2, CYP3, CYP4 and CYP5 were also studied in various allelic combinations. They show some interactions between them or with CYC1 locus but these interactions are different and less pronounced than those involving loci CYP1 and CYC1.     

Iso-cytochrome c species from baker''s yeast. Analysis of their circular-dichroism spectra.

The circular-dichroism spectra of baker's-yeast iso-1- (methylated and unmethylated forms) and iso-2-cytochrome c species were examined between 200 and 600nm. In the visible region the yeast haemoproteins have characteristics nearly indistinguishable from those of horse heart cytochrome c. From the spectra in the u.v. region the latter appears, however, to be more helical. It is proposed that the likely element of non-helical structure in iso-1-cytochrome c is residues 62-70.     

Structural insights into the conformation and oligomerization of E2~ubiquitin conjugates

Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes, including protein quality control, DNA repair, endocytosis, and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the C-terminus of Ub and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent "backside" interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers, and the mechanisms of ubiquitination. We present a new 2.35 ? crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the "infinite spiral" helical arrangement of the only previously reported structure of an oligomeric conjugate. Our structure also differs in intraconjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intraconjugate relative E2-Ub orientations. We delineate a common intraconjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3s, which have important consequences for the ubiquitination process.     

CYP3 a likely candidate for the structural gene of ISO-2 cytochrome C in Saccaromyces cerevisiae.

Mutations specific for iso-2 cytochrome c were obtained in strains bearing a deletion of the structural gene of iso-1-cytochrome c. In this genetic context the mutations entail an inability to grow on glycerol. One of these mutants was shown to have a modified iso-2-cytochrome c as witnessed by its lack of stability and modified chromatographic behaviour. Genetic studies showed the mutations to be allelic to the mutation cyp3-15 previously identified by Clavilier $\text{et al.}$ (1) as a specific enhancer of iso-2-cytochrome c synthesis. The simplesthypothesis to explain the results is that the CYP3 locus is the structural gene for iso-2-cytochrome c.     

Further investigations regarding the role of trimethyllysine for cytochrome c uptake into mitochondria.

1. A mutant of the iso-1-cytochrome c gene from Saccharomyces cerevisiae has been constructed which contains an Arg codon, replacing the normal trimethylated Lys at position 77. 2. This mutated gene was cloned into a pGem 1 vector and used for the in vitro translation of yeast iso-1-cytochrome c. 3. Utilizing an in vitro mitochondria binding assay, it was found that the mutant cytochrome c could transverse the yeast mitochondrial membrane, however the amount of protein incorporated was 3-fold less that of the trimethylated wild type. 4. Omission of the protein methyltransferase from assays containing the wild type cytochrome c caused only a slight reduction (15%) in the amount of protein incorporated. 5. These results suggest while the lysine residue 77 of apocytochrome c is important for mitochondria uptake, the methylation of this residue seems to play a relatively minor role.     

Demonstration of several independent loci involved in the synthesis of iso-2-cytochrome c in yeast

Summary This study concerns the chromosomal genes controlling the synthesis of cytochrome c in yeast. In the wild type there are two molecular species of cytochrome c : iso-1 (major from) and iso-2 (minor form) which differ in many positions of their amino-acid sequence. A mutation, CY1cy1-1, in the structural gene for iso-1, leads to iso-1 deficiency, while retaining a normal albeit small amount of iso-2-cytochrome c.The cyI-1 mutant does not grow on DL-lactate as sole carbon source, while the wild type does. This property was used for selecting cytochrome c rich revertants (CYT) from cytochrome c deficient strains cy1-1; ca 200 revertants were isolated after extensive nitrous acid mutagenesis from a haploid cy1-1 strain or from a diploid cy1-1/cy1-1 strain and ca 30 of them were analyzed genetically and biochemically. The cytochrome c of seven (CYT) revertants was extracted and characterized; none of them contained iso-1-cytochrome c, but all contained large amount of iso-2-cytochrome csufficient to compensate for the deficiency. It was concluded that none of the revertants resulted from back mutation of cy1-1 and that the cy1-1 mutation is a deletion or some other irreversible aberration. These conclusions were corroborated by genetic analysis. It was shown that every reversion is due to a chromosomal mutation segregating as a single gene. Five unlinked gene loci, CY2A, CY2B, CY2C, CY2D, CY2E, were uncovered in this way. None of them were linked to the CY1 locus. Revertants selected in the diploid strain were dominant or semi-dominant while those selected in the haploid strain were recessive. To the first class belong alleles at loci CY2A, CY2B, CY2C, while to the latter belong alleles at loci CY2D and CY2E.Five unlinked loci are implicated in iso-2-cytochrome c synthesis. Mutations selected at these loci act as suppressors of cytochrome c deficiency caused by a deletion of the CY1 locus. In fact the muations do not restore the synthesis of the deficient protein (iso-1-cytochrome c), but increase the synthesis of an another protein, structurally alike (iso-2-cytochrome c), and having very similar if not identical physiological activity. We propose the term of compensator genes to define this type of mutations. We discuss some possible mechanisms to explain the rarity of compensator mutations and the hypothesis that the locus CY2A could correspond not only to the regulatory gene for iso-2-cytochrome c but also to the structural one.     

Structure determination and analysis of yeast iso-2-cytochrome c and a composite mutant protein.

As part of a study of protein folding and stability, the three-dimensional structures of yeast iso-2-cytochrome c and a composite protein (B-2036) composed of primary sequences of both iso-1 and iso-2-cytochromes c have been solved to 1.9 A and 1.95 A resolutions, respectively, using X-ray diffraction techniques. The sequences of iso-1 and iso-2-cytochrome c share approximately 84% identity and the B-2036 composite protein has residues 15 to 63 from iso-2-cytochrome c with the rest being derived form the iso-1 protein. Comparison of these structures reveals that amino acid substitutions result in alterations in the details of intramolecular interactions. Specifically, the substitution Leu98Met results in the filling of an internal cavity present in iso-1-cytochrome c. Further substitutions of Val20Ile and Cys102Ala alter the packing of secondary structure elements in the iso-2 protein. Blending the isozymic amino acid sequences in this latter area results in the expansion of the volume of an internal cavity in the B-2036 structure to relieve a steric clash between Ile20 and Cys102. Modification of hydrogen bonding and protein packing without disrupting the protein fold is illustrated by the His26Asn and Asn63Ser substitutions between iso-1 and iso-2-cytochromes c. Alternatively, a change in main-chain fold is observed at Gly37 apparently due to a remote amino acid substitution. Further structural changes occur at Phe82 and the amino terminus where a four residue extension is present in yeast iso-2-cytochrome c. An additional comparison with all other eukaryotic cytochrome c structures determined to date is presented, along with an analysis of conserved water molecules. Also determined are the midpoint reduction potentials of iso-2 and B-2036 cytochromes c using direct electrochemistry. The values obtained are 286 and 288 mV, respectively, indicating that the amino acid substitutions present have had only a small impact on the heme reduction potential in comparison to iso-1-cytochrome c, which has a reduction potential of 290 mV.     

Ubiquitin in motion: structural studies of the ubiquitin-conjugating enzyme∼ubiquitin conjugate

Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2～Ub conjugate. Therefore, full characterization of the structure and dynamics of E2～Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2～Ub conjugates of two human enzymes, UbcH5c～Ub and Ubc13～Ub, in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c～Ub conjugate populates an array of extended conformations, and the population of Ubc13～Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2～Ub species and thus provide insight into the diverse E2～Ub protein interactome, particularly with regard to interaction with Ub ligases.     

Cytochrome c from Schizosaccharomyces pombe. 1. Purification, spectral properties, and amino-acid composition.

Cytochrome c from the fission yeast Schizosaccharomyces pombe has been purified. Its chromatographic and spectral properties are reported and compared to those of iso-1-cytochrome c from baker's yeast; the amino-acid composition is described. Schiz. pombe cytochrome c has a much lower affinity for Amberlite IRP64 than Sacch. cerevisiae iso-1-cytochrome c. Its alpha absorption band splits into three maxima (calpha1, calpha2, and calpha3) at -190 degrees C; this is unusual in yeasts, as shown by the low-temperature whole-cell absorption spectra which were examined in various yeast genera, species, and strains. A minor component can be separated by Amberlite chromatography. It exhibits the same low-temperature splitting of the alpha absorption band as the main fraction and it has a similar amino-acid composition with a notable exception: it is an unmethylated form of the cytochrome.     

A study of the influence of the hydrophobic core residues of yeast iso-2-cytochrome c on phosphate binding: a probe of the hydrophobic core-surface charge interactions

To gain insight into the role of hydrophobic core-surface charge interactions in stabilizing cytochrome c, we investigated the influence of hydrophobic core residues on phosphate binding by mutating residues in yeast iso-2-cytochrome c to those corresponding to iso-1-cytochrome c in various combinations. Heat transition of ultraviolet CD was followed as a function of pH in the presence and absence of phosphate. Thermodynamic parameters were deduced. It was found that the I20V/V43A/M98L mutation in the hydrophobic core, whose locations are remote from the putative phosphate sites, modulates phosphate interactions. The modulation is pH dependent. The I20V/M98L and V43A mutation effects are nonadditive. The results lead to a model analogous to that of Tsao, Evans, and Wennerstrom, where a domain associated with the ordered hydrophobic core is sensitive to the fields generated by the surface charges. Such an explanation would be in accord with the observed difference in thermal stability between iso-2 and horse cytochromes c.     

Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13

UBC13 is the only known E2 ubiquitin (Ub)-conjugating enzyme that produces Lys-63-linked Ub chain with its cofactor E2 variant UEV1a or MMS2. Lys-63-linked ubiquitination is crucial for recruitment of DNA repair and damage response molecules to sites of DNA double-strand breaks (DSBs). A deubiquitinating enzyme OTUB1 suppresses Lys-63-linked ubiquitination of chromatin surrounding DSBs by binding UBC13 to inhibit its E2 activity independently of the isopeptidase activity. OTUB1 strongly suppresses UBC13-dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro. The mechanism of this non-canonical OTUB1-mediated inhibition of ubiquitination remains to be elucidated. Furthermore, the atomic level information of the interaction between human OTUB1 and UBC13 has not been reported. Here, we determined the crystal structure of human OTUB1 in complex with human UBC13 and MMS2 at 3.15 Å resolution. The presented atomic-level interactions were confirmed by surface-plasmon resonance spectroscopy with structure-based mutagenesis. The designed OTUB1 mutants cannot inhibit Lys-63-linked Ub chain formation in vitro and histone ubiquitination and 53BP1 assembly around DSB sites in vivo. Finally, we propose a model for how capping of di-Ub by the OTUB1-UBC13-MMS2/UEV1a complex efficiently inhibits Lys-63-linked tri-Ub formation.     

A SnapShot of Ubiquitin Chain Elongation: LYSINE 48-TETRA-UBIQUITIN SLOWS DOWN UBIQUITINATION*

We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1-βTrCP F-box protein (SCF^βTrCP) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF^βTrCP formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using “elongation intermediates” containing β-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF^βTrCP/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.     

Interaction of yeast iso-1-cytochrome c with cytochrome c peroxidase investigated by [15N, 1H] heteronuclear NMR spectroscopy

The interaction of yeast iso-1-cytochrome c with its physiological redox partner cytochrome c peroxidase has been investigated using heteronuclear NMR techniques. Chemical shift perturbations for both 15N and 1H nuclei arising from the interaction of isotopically enriched 15N cytochrome c with cytochrome c peroxidase have been observed. For the diamagnetic, ferrous cytochrome c, 34 amides are affected by binding, corresponding to residues at the front face of the protein and in agreement with the interface observed in the 1:1 crystal structure of the complex. In contrast, for the paramagnetic, ferric protein, 56 amides are affected, corresponding to residues both at the front and toward the rear of the protein. In addition, the chemical shift perturbations were larger for the ferric protein. Using experimentally observed pseudocontact shifts the magnetic susceptibility tensor of yeast iso-1-cytochrome c in both the free and bound forms has been calculated with HN nuclei as inputs. In contrast to an earlier study, the results indicate that there is no change in the geometry of the magnetic axes for cytochrome c upon binding to cytochrome c peroxidase. This leads us to conclude that the additional effects observed for the ferric protein arise either from a difference in binding mode or from the more flexible overall structure causing a transmittance effect upon binding.     

Activation of the Slx5-Slx8 ubiquitin ligase by poly-small ubiquitin-like modifier conjugates

Protein sumoylation is a regulated process that is important for the health of human and yeast cells. In budding yeast, a subset of sumoylated proteins is targeted for ubiquitination by a conserved heterodimeric ubiquitin (Ub) ligase, Slx5-Slx8, which is needed to suppress the accumulation of high molecular weight small ubiquitin-like modifier (SUMO) conjugates. Structure-function analysis indicates that the Slx5-Slx8 complex contains multiple SUMO-binding domains that are collectively required for in vivo function. To determine the specificity of Slx5-Slx8, we assayed its Ub ligase activity using sumoylated Siz2 as an in vitro substrate. In contrast to unsumoylated or multisumoylated Siz2, substrates containing poly-SUMO conjugates were efficiently ubiquitinated by Slx5-Slx8. Although Siz2 itself was ubiquitinated, the bulk of the Ub was conjugated to SUMO residues. Slx5-Slx8 primarily mono-ubiquitinated the N-terminal SUMO moiety of the chain. These data indicate that the Slx5-Slx8 Ub ligase is stimulated by poly-SUMO conjugates and that it can ubiquitinate a poly-SUMO chain.