c-Type Cytochrome Assembly in Saccharomyces cerevisiae: A Key Residue for Apocytochrome c1/Lyase Interaction

The electron transport chains in the membranes of bacteria and organelles generate proton-motive force essential for ATP production. The c-type cytochromes, defined by the covalent attachment of heme to a CXXCH motif, are key electron carriers in these energy-transducing membranes. In mitochondria, cytochromes c and c₁ are assembled by the cytochrome c heme lyases (CCHL and CC₁HL) and by Cyc2p, a putative redox protein. A cytochrome c₁ mutant with a CAPCH heme-binding site instead of the wild-type CAACH is strictly dependent upon Cyc2p for assembly. In this context, we found that overexpression of CC₁HL, as well as mutations of the proline in the CAPCH site to H, L, S, or T residues, can bypass the absence of Cyc2p. The P mutation was postulated to shift the CXXCH motif to an oxidized form, which must be reduced in a Cyc2p-dependent reaction before heme ligation. However, measurement of the redox midpoint potential of apocytochrome c₁ indicates that neither the P nor the T residues impact the thermodynamic propensity of the CXXCH motif to occur in a disulfide vs. dithiol form. We show instead that the identity of the second intervening residue in the CXXCH motif is key in determining the CCHL-dependent vs. CC₁HL-dependent assembly of holocytochrome c₁. We also provide evidence that Cyc2p is dedicated to the CCHL pathway and is not required for the CC₁HL-dependent assembly of cytochrome c₁.THE c-type cytochromes, also referred to as cytochrome c, represent a universal class of heme-containing proteins that function as electron carriers in the energy-transducing pathways of bacteria, plastids, and mitochondria (Thöny-Meyer 1997; Nakamoto et al. 2000; Bonnard et al. 2010). Because cytochromes c carry a heme covalently attached to a CXXCH motif, they constitute an attractive object of study to address the question of cofactor protein assembly. The biochemical requirements for cytochrome c assembly were deduced from in vivo and in vitro studies, and the conclusion is that both apocytochromes c and heme are transported independently across at least one biological membrane and maintained as reduced prior to catalysis of the heme attachment reaction (Allen et al. 2003; Hamel et al. 2009; Kranz et al. 2009; Sanders et al. 2010). Bacterial cytochromes c are assembled in the periplasmic space, a compartment where cysteine pairs in proteins form disulfide bonds in reactions catalyzed by dedicated enzymes (Inaba 2009; Kadokura and Beckwith 2010). The current thinking holds that a c-type apocytochrome is a substrate of the disulfide bond-forming pathway, which introduces an intramolecular disulfide between the two cysteines of the CXXCH sequence (Allen et al. 2003; Sanders et al. 2010). This disulfide needs to be reduced to a dithiol to provide free sulfhydryls for the heme ligation. Consistent with this view is the fact that groups of specific oxido-reductases that constitute a transmembrane dithiol-disulfide relay from the cytosol to the periplasmic space have been shown to function as c-type cytochrome assembly factors (Allen et al. 2003; Kadokura et al. 2003; Mapller and Hederstedt 2006; Sanders et al. 2010). The proposal that the components of this pathway control the in vivo redox status of the CXXCH sulfhydryls has been inferred from the presence of motifs in their protein sequences that are consistent with a function in redox chemistry and also from the demonstration that their recombinant forms participate in dithiol–disulfide exchange reactions (Monika et al. 1997; Setterdahl et al. 2000). Moreover, the ability of exogenous thiol compounds to bypass the lack of these factors in vivo substantiates the view that the redox components have a disulfide-reducing activity in the pathway (e.g., Sambongi and Ferguson 1994; Fabianek et al. 1998; Beckett et al. 2000; Deshmukh et al. 2000; Bardischewsky and Friedrich 2001; Erlendsson and Hederstedt 2002; Erlendsson et al. 2003; Feissner et al. 2005; Turkarslan et al. 2008).While the role of these pathways is well established in bacteria, much less is known about the components that catalyze thiol/disulfide chemistry in the mitochondrial intermembrane space (IMS), which is topologically equivalent to the bacterial periplasm. By analogy with the bacterial pathways, the participation of redox-active factors that catalyze thiol formation is expected, as the mitochondrial IMS houses two c-type cytochromes, the soluble cytochrome c and the membrane-bound cytochrome c₁, both of which function in respiration. In fungi, heme attachment to apocytochromes c and c₁ is dependent upon the IMS resident cytochrome c and c₁ heme lyases, CCHL and CC₁HL, although the exact role of these lyases in the assembly process is still unclear (Dumont et al. 1987; Zollner et al. 1992). Conversion of apocytochrome to holocytochrome c depends only on CCHL, while apocytochrome c₁ can be acted upon by both CCHL and CC₁HL (Matner and Sherman 1982; Dumont et al. 1987; Stuart et al. 1990; Zollner et al. 1992; Bernard et al. 2003). In animals, apoforms of cytochromes c and c₁ are assembled by a unique heme lyase, HCCS, which carries both the CCHL and CC₁HL activities (Prakash et al. 2002; Schwarz and Cox 2002; Bernard et al. 2003).Cyc2p, a component first described as a mitochondrial biogenesis factor in yeast (Matner and Sherman 1982; Dumont et al. 1993; Pearce et al. 1998; Sanchez et al. 2001), was recently rediscovered in the context of cytochrome c₁ maturation (Bernard et al. 2003). Cyc2p is located at the mitochondrial inner membrane with its C-terminal domain containing a non-covalently bound FAD exposed to the IMS (Bernard et al. 2005). A redox function for Cyc2p is likely based on the finding that a recombinant form of the molecule exhibits a NAD(P)H-dependent reductase activity (Bernard et al. 2005). However, as Cyc2p activity is not essential for the maturation process, a functional redundancy was postulated based on the fact that a cyc2-null mutant still assembles holoforms of cytochromes c and c₁ (Bernard et al. 2005). The absolute requirement of Cyc2p was revealed via genetic analysis of the cyc2-null cyt1-34 combination that displays a synthetic respiratory-deficient phenotype with loss of holocytochrome c₁ assembly (Bernard et al. 2005). The cyt1-34 mutation maps to the gene encoding cytochrome c₁ and results in a CAPCH heme-binding site replacing the wild-type CAACH site (Bernard et al. 2005). The synthetic interaction is specific for the cyt1-34 allele carrying the A-to-P mutation and is not observed in a cyc2-null cyt1-48 strain carrying an A-to-D mutation at the heme-binding site of apocytochrome c₁ (Bernard et al. 2005). The fact that Cyc2p becomes essential when the cytochrome c₁ heme-binding site carries an A-to-P mutation suggests that the CXXCH motif could be the target of Cyc2p action in vivo. One possible interpretation for this observation is that the P residue alters the reactivity of the cysteinyl thiols to redox chemistry so that the apocytochrome c₁ CAPCH heme-binding site occurs in an oxidized (disulfide) form, which must be reduced in a Cyc2p-dependent reaction before heme attachment can occur.In this article, we have undertaken a genetic approach to elucidate this pathway and searched for suppressors that alleviate the respiratory deficiency of the cyc2-null cyt1-34 strain. Either overexpression of CC₁HL or replacement of the P mutation in the heme-binding site by H, L, S, or T residues restore the assembly of holocytochrome c₁. In vitro measurement of redox potential of apoforms of CA(A/P/T)CH cytochrome c₁ indicates that there is no change in the thermodynamic stability of the disulfide at the CXXCH motif that could account for the Cyc2p-dependent assembly of cytochrome c₁. Genetic studies reveal that the replacement of the second A residue at the CAACH motif by H, L, P, S, and T residues is key in determining the conversion of apocytochrome c₁ to its corresponding holoform via the CCHL and/or CC₁HL-dependent pathway. We also demonstrate that Cyc2p is a component dedicated to the CCHL pathway and is not required for the CC₁HL-dependent assembly of cytochrome c₁. We propose that the CAPCH cytochrome c₁ is strictly dependent upon CCHL and Cyc2p for its assembly but becomes a substrate of CC₁HL upon overexpression of CC₁HL or in the presence of H, L, S, or T mutations.     

The number of neurophysins in the rat. Influence of the concentration of Bromophenol Blue, used as a tracking dye, on the resolution of proteins by polyacrylamide-gel electrophoresis

1. The concentration of Bromophenol Blue used as tracking dye in polyacrylamide-gel electrophoresis affected the resolution of rat neurophysins. 2. A final dye concentration of 1mug/ml in the tris-glycine running buffer was found to give the best results. 3. The presence of two major and one minor neurophysin(s) in the rat was confirmed. 4. The two major proteins were found to re-run as single discrete bands, which had the same mobilities in the absence of dye and different mobilities in its presence.     

Donor T cell activation initiates small bowel allograft rejection through an IFN-gamma-inducible protein-10-dependent mechanism

The poor success in controlling small bowel (SB) allograft rejection is partially attributed to the unique immune environment in the donor intestine. We hypothesized that Ag-induced activation of donor-derived T cells contributes to the initiation of SB allograft rejection. To address the role of donor T cell activation in SB transplantation, SB grafts from DO11.10 TCR transgenic mice (BALB/c, H-2L(d+)) were transplanted into BALB/c (isografts), or single class I MHC-mismatched (L(d)-deficient) BALB/c H-2(dm2) (dm2, H-2L(d-)) mutant mice (allografts). Graft survival was followed after injection of control or antigenic OVA(323-339) peptide. Eighty percent of SB allografts developed severe rejection in mice treated with antigenic peptide, whereas <20% of allografts were rejected in mice treated with control peptide (p < 0.05). Isografts survived >30 days regardless of OVA(323-339) administration. Activation of donor T cells increased intragraft expression of proinflammatory cytokine (IFN-gamma) and CXC chemokine IFN-gamma-inducible protein-10 mRNA and enhanced activation and accumulation of host NK and T cells in SB allografts. Treatment of mice with neutralizing anti-IFN-gamma-inducible protein-10 mAb increased SB allograft survival in Ag-treated mice (67%; p < 0.05) and reduced accumulation of host T cells and NK cells in the lamina propria but not mesenteric lymph nodes. These results suggest that activation of donor T cells after SB allotransplantation induces production of a Th1-like profile of cytokines and CXC chemokines that enhance infiltration of host T cells and NK cells in SB allografts. Blocking this pathway may be of therapeutic value in controlling SB allograft rejection.     

Elimination of chromosomes in Hordeum vulgare x H. bulbosum crosses at mitosis and interphase involves micronucleus formation and progressive heterochromatinization

Uniparental chromosome elimination occurs in several interspecific hybrids of plants. We studied the mechanism underlying selective elimination of the paternal chromosomes during the development of Hordeum vulgare x H. bulbosum hybrid embryos that is restricted to an early stage of development. In almost all embryos most of the H. bulbosum chromatin undergoes a fast rate of elimination within nine days after pollination. There are differences in the mitotic behaviour between the parental chromosomes, with H. bulbosum chromatids segregating asymmetrically at anaphase. We provide evidence for a chromosome elimination pathway that involves the formation of nuclear extrusions during interphase in addition to postmitotically formed micronuclei. The chromatin structure of nuclei and micronuclei differs and heterochromatinization and disintegration of the nuclear envelope of micronuclei are the final steps of chromosome elimination.