Research Plan
ASSEMBLY PATHWAYS OF PLASTID AND MITOCHONDRIAL C-TYPE CYTOCHROMES
The c-type cytochromes are an attractive model for the study of cofactor-protein assembly
The c-type cytochromes are universal hemeproteins that function typically as electron carriers on the pside of energy-transducing membrane systems. They are distinguished by the occurrence of heme covalently
attached to the polypeptide via two (or very rarely one) thioether linkages between the vinyl groups of heme
and the cysteines sulfhydryls in the apocytochrome. A CXXCH2 sequence in the apocytochrome provides the
thiols for formation of the thioether bonds, and the imidazole of the histidine residue serves as one of the axial
ligands of the heme. The c-type cytochromes have been the focus of intense investigation from mechanism to
structure but the details of their maturation remain poorly understood.
1
Remarkably, three distinct assembly pathways, referred to as systems I, II and III, have emerged
through genetic analysis of c-type cytochrome maturation in bacteria, chloroplasts and mitochondria (#8 of my
biosketch), an unexpected finding for what appears on the surface to be a rather simple chemical reaction (i. e.
addition of apocytochrome cysteine thiols to the vinyls of heme). Each system can be distinguished on the
basis of sequence relationship of specific assembly factors. Yet, there is an underlying assumption that the
three systems are united by common biochemical requirements for holocytochrome c biogenesisa. Indeed,
regardless of the experimental system under study, heme and apocytochromes need to be transported
independently to the p-side of the energy-transducing membrane and maintained in a reduced form prior to
catalysis of heme attachment (see Fig.1).
While considerable detail is known of bacterial cytochrome c maturation, the biogenesis of c-type
cytochrome in organelles has remained so far under-investigated. For instance, the existence of heme delivery
routes and the operation of a thioreduction pathway for the supply of reductants to the site of cytochrome c
assembly which have been described in bacteria are completely unknown in mitochondria and now only
emerging in plastids. Cytochromes c have received renewed attention with the discovery that, beside their wellestablished participation in redox chemistry, they can also be recruited for other functions such as signaling in
mitochondria and plastids. It is therefore conceivable that there is a lot more to be discovered in the function
and biogenesis of these versatile molecules. Indeed, an intriguing feature is that plastids and mitochondria
harbor analogous c-type cytochromes in terms of function but distinct in terms of mechanisms of assembly. My
long-term goal is to elaborate the biochemistry of cytochrome c maturation in organelles by identifying all the
components involved in the assembly process and deduce what activity each component is responsible for. In
the next five years, I will approach the following : 1) Exploration of the plastid redox pathway, its components
and their sequence of action and identification of other CCS factors 2) Functional analysis of pioneer
flavoprotein Cyc2p and discovery of novel mitochondrial assembly factors and 3) investigation of general thiol
chemistry in thylakoid lumen and mitochondrial IMS.
Nine components define the maturation pathway for plastid c-type cytochromes
The pathway in plastids was initially discovered by the Merchant laboratory using Chlamydomonas as
an experimental system. In this green alga, one plastid locus, ccsA3 and 6 nuclear loci (CCS1 to CCS6) are
required for the assembly of membrane-bound cyt f and soluble cyt c6 in the plastid lumen (#8). However, the
genetic screens have not been saturated and more loci could be revealed. At present, only ccsA and CCS1 are
known at the molecular level and they encode novel membrane polytopic proteins. I have proposed that CcsA
and Ccs1 act together in a heme delivery pathway from stroma to lumen and postulated that they operate with
genetically-defined Ccs2, Ccs3 and Ccs4 factors through a 200 kDa complex that constitutes a cytochrome c
assembly machinery in the thylakoid membranes (#10,11). The characterization of assembly factors through
1
The p-side corresponds to the plastid lumen, mitochondrial intermembrane space, or bacterial periplasm. The n-side is
the plastid stroma, mitochondrial matrix or the bacterial cytoplasm.
2 Single letter amino-acid code where X is any amino-acid.
3 ccs for cytochrome c synthesis
1
Patrice P. HAMEL, Ph.D.
Research Plan
ASSEMBLY PATHWAYS OF PLASTID AND MITOCHONDRIAL C-TYPE CYTOCHROMES
the cloning of CCS loci and the purification and resolution of the CCS complex components still remain a
priority of my research plan.
Interestingly, in the bacteria where the CCS pathway operates, only 4 components, namely CcsA,
Ccs1, CcdA and CcsX/HCF164 are known from genetics. CcdA and CcsX/HCF164 are components of a redox
subpathway involved in the transfer of reducing equivalents for the maintenance of reduced apocytochrome
sulfhydryls prior to the ligation of heme. Genes encoding homologs of CcdA and CcsX are present in the
nuclear genome of Chlamydomonas but they do not correspond to any of the CCS2 to CCS6 loci (#14).
Nevertheless, our recent work in the plant model Arabidopsis supports the involvement of CcdA in a plastid
thioreduction pathway in cytochrome c maturation (# 14). Our current view is that biogenesis of plastid c-type
cytochromes depends upon the activity of 9 components, CcsA, Ccs1 to Ccs6, CCDA and CcsX/HCF164. A
preliminary functional assessment of CCS4 and CCS5 genes in redox chemistry is based on the observation
that ccs4 and ccs5 mutants can be rescued for cytochrome c deficiency by exogenous reduced thiol
compounds (#14). It is conceivable that, due to the compartmentalization of the thylakoid lumen, the redox
pathway evolved in plastids relies on several players (at least CCDA, HCF164, Ccs4 and Ccs5) and is
therefore more complex than its bacterial counterpart. We have made progress toward the identification of both
CCS4 and CCS5 genes and we predict that they encode novel redox components.
A novel flavoprotein and a candidate cytochrome c assembly complex in mitochondria
As my appreciation of cytochrome biogenesis mechanisms developed through the study of the plastid
pathway, I became curious about the apparent unique features of the fungal and animal mitochondrial
pathway, where despite exhaustive genetic analyses, only two factors have been identified: the CCHL and
related CC1HL, responsible presumably for thioether bond formation in soluble cyt c and membrane-bound cyt
c1, respectively. However, the biochemistry of these proteins has not been detailed thoroughly and biochemical
proof of a direct enzymatic function of purified CCHLs is still lacking.
While bacterial and plastid cytochrome c assembly factors show broad apoprotein substrate specificity,
the prevailing dogma was that CCHL and CC1HL show non-overlapping substrate specificity for their
respective apocytochrome. We showed, in the S. cerevisiae model for the mitochondrial pathway, that the
CCHL does display intrinsic weak activity towards its non-cognate substrate apocyt c1, while CC1HL activity is
strictly specific for apocyt c1 (#12). The intrinsic weak activity of CCHL towards apocyt c1 can be enhanced by
mutations in either CCHL (the enzyme) or in the apocyt c1 substrate (e.g. mutations at the heme binding
CXXCH motif), which provides for the first time definitive genetic evidence for an interaction between the CCHL
and its cytochrome substrate (#12).
Two key problems concerning mitochondrial cytochrome c biogenesis are raised in the context of 1) the
known biochemical requirements for compartmentalized holocytochrome c formation and 2) the extraordinary
complexity of the plastid pathway. How is heme supplied from the matrix to the intermembrane space (Fig.1)?
And, how are the substrates (cysteinyl thiols and heme iron) in the IMS maintained in the reduced form? That a
specific mechanism for substrate reduction must exist is indicated by the requirement for exogenous redox
cofactors, both pyridine and flavin nucleotides, for the isolated in organello reaction studies. We have made
progress in the understanding of these problems with the discovery of Cyc2p, a novel putative redox
component and a candidate CCHL-containing complex in mitochondria.
Cyc2p was isolated as a partner of CCHL (#12). This molecule had been characterized previously as a
“general” factor in the biogenesis of mitochondria, but its function is still obscure. Our unpublished work
suggests a direct role for Cyc2p in the redox chemistry of the cytochrome assembly pathway based on: 1) the
presence of a previously unrecognized FAD binding domain in the C-terminal portion of the protein that binds
FAD and faces the intermembrane space (where cytochrome biogenesis is completed), and 2) a synthetic
interaction between Δcyc2 and a mutation in the heme binding site of cyt c1 (CAPCH instead of CAACH in wild
type), which suggests that the site of action of Cyc2p is the heme binding site. We imagine that the A to P
2
Patrice P. HAMEL, Ph.D.
Research Plan
ASSEMBLY PATHWAYS OF PLASTID AND MITOCHONDRIAL C-TYPE CYTOCHROMES
mutation alters the reactivity of the cysteinyl thiols to the extent that Cyc2p action is now required. An attractive
hypothesis is that the cysteines of the CAPCH motif have the propensity to form an intra-molecular disulfide
bond that is resolved by Cyc2p.This proposal is consistent with a role of Cyc2p as a disulfide reductase and
solidifies its placement in the redox pathway for cyt c assembly. Biochemical analysis of Cyc2p including
measurement of the redox activity in vitro and assessment of redox status of CAPCH in vivo is the next
obvious step in order to delineate the activity of this unique assembly factor. Given that the absence of Cyc2p
does not lead to a complete deficiency in c-type cytochrome suggests functional redundancy, which could be
revealed through a genetic screen for overlapping function with Cyc2p. This will be achieved by using a high
throughput approach at UCSF (in the Jonathan Weissman laboratory, a leader in the field of redox components
in the ER). Novel components controlling IMS redox chemistry are expected to be unveiled through such a
screen.
Using the BN-PAGE technique, we discovered recently that CCHL occurs in a 300 kDa complex in
mitochondrial inner membranes that does not contain CC1HL and Cyc2p (unpublished). It is formally possible
that the 300 kDa species consists of a multimeric form of CCHL, but it is more likely that there are other
proteins involved in cytochrome assembly (similarly to the CCS complex in plastid) that have not been
identified genetically because of overlapping / redundant functions. We are now in the position to identify
interacting components by resolution of the different constituents of the CCHL complex using affinity
purification method and mass spectrometry. Once their identity is established, functional analysis will be
carried out to define their participation in cytochrome maturation and other mitochondrial processes. If motifs
like those involved in thiol-disulfide chemistry or heme binding are present, specific biochemical test can be
developed to establish their biochemical activity.
Crystallization of CCHL and the FAD binding domain of Cyc2p is now in progress (collaboration with
Drs. Guiard and Quevillon-Cheruel, University of Paris-Sud) and we anticipate that novel insights into the
function of these proteins will be revealed from their three dimensional structures.
Significance of thiol metabolism pathways in the mitochondrial IMS and thylakoid lumen
The studies of bacterial cytochrome c biogenesis resulted in the unanticipated elaboration of
periplasmic thiol metabolizing pathways, which are required not only for cyt c maturation but also for the
maintenance of a variety of cysteine-containing protein substrates. For instance, the thiol-reducing branch in
the periplasm maintains the reduction state of protein disulfide isomerases whose activity is important for
shuffling the S-S bonds in proteins that exit the oxidative folding pathway. The components of thiol
metabolizing pathways in the corresponding organelle compartments (thylakoid lumen and mitochondrial
intermembrane space) are not known. It is possible that redox cyt c assembly factors (like CcdA and Cyc2p for
instance) in chloroplasts and mitochondria function also in the maintenance of thiol-disulfide status of other
proteins. The occurrence of regulated disulfide bonded proteins in the lumen, like Rieske FeS and in the IMS
like the small Tims of the import machinery suggests that catalysts of disulfide bond formation, yet to be
discovered, exist in the organelles. My long-term goal is to examine if general thiol chemistry also takes place
in the plastid lumen and mitochondrial IMS.
The novelty of the assembly factors in plastids and the emergence of redox chemistry in the
mitochondrial system demonstrates the potential of the project for new discovery in the area of membrane
biogenesis. The recent finding that loss of CCHL in humans results in a neurodegenerative disease with
cardiomyopathic manifestations speaks also to the broad relevance and impact of the future discoveries.
Figure 1: Apocytochrome c and heme need to be transported
independently to the p-side and maintained reduced prior to the
ligation of heme. How heme is transported from n-side (where it is
synthesized) to p-side is not known in organelles. Redox
components involved in organellar cyt c assembly are beginning to
emerge. General thiol chemistry on the p-side of the membrane has
not been explored in organelles.
3
Patrice P. HAMEL, Ph.D.
Research Plan
ASSEMBLY PATHWAYS OF PLASTID AND MITOCHONDRIAL C-TYPE CYTOCHROMES
4
Patrice P. HAMEL, Ph.D.