Bernard Guiard

The proteome of Saccharomyces cerevisiae mitochondria

We performed a comprehensive approach to determine the proteome of Saccharomyces cerevisiae mitochondria. The proteins of highly pure yeast mitochondria were separated by several independent methods and analyzed by tandem MS. From >20 million MS spectra, 750 different proteins were identified, indicating an involvement of mitochondria in numerous cellular processes. All known components of the oxidative phosphorylation machinery, the tricarboxylic acid cycle, and the stable mitochondria-encoded proteins were found. Based on the mitochondrial proteins described in the literature so far, we calculate that the identified proteins represent ≈90% of all mitochondrial proteins. The function of a quarter of the identified proteins is unknown. The mitochondrial proteome will provide an important database for the analysis of new mitochondrial and mitochondria-associated functions and the characterization of mitochondrial diseases.

Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins

Mitochondria import nuclear‐encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.

Mitochondrial Presequence Translocase: Switching between TOM Tethering and Motor Recruitment Involves Tim21 and Tim17

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) operates at a central junction of protein import. It accepts preproteins from the outer membrane TOM complex and directs them to inner membrane insertion or, in cooperation with the presequence translocase-associated motor (PAM), to the matrix. Little is known of how the TIM23 complex coordinates these tasks. We have identified Tim21 (YGR033c) that interacts with the TOM complex. Tim21 is specific for a TIM23 form that cooperates with TOM and promotes inner membrane insertion. Protein translocation into the matrix requires a switch to a Tim21-free, PAM bound presequence translocase. Tim17 is crucial for the switch by performing two separable functions: promotion of inner membrane insertion and binding of Pam18 to form the functional TIM-PAM complex. Thus, the presequence translocase is not a static complex but switches between TOM tethering and PAM binding in a reaction cycle involving Tim21 and Tim17.

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70–Tim44

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70–Tim44 driving system. By blue native electrophoresis, we identify an ∼90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence‐containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

The ABC transporter Atm1p is required for mitochondrial iron homeostasis

The function of the ABC transporter Atm1p located in the mitochondrial inner membrane is not yet known. To study its cellular role, we analyzed a mutant in which ATM1 was disrupted. Δatm1 cells are deficient in the holoforms, but not the apoforms of heme‐carrying proteins both within and outside mitochondria, yet both synthesis and transport of heme are functional. Δatm1 cells are hypersensitive for growth in the presence of oxidative reagents, and they contain increased levels of the antioxidant glutathione, in particular of its oxidized form. Mitochondria deficient in Atm1p accumulate 30‐fold higher levels of free iron as compared to wild‐type organelles, i.e. three‐fold more than mitochondria deficient in frataxin, the protein mutated in Friedreichs ataxia. The increased mitochondrial iron content may be causative of the oxidative damage of heme‐containing proteins in Δatm1 cells. Our data assign an important function to Atm1p in mitochondrial iron homeostasis.

Dissecting Membrane Insertion of Mitochondrial β-Barrel Proteins

Communication of mitochondria with the rest of the cell requires beta-barrel proteins of the outer membrane. All beta-barrel proteins are synthesized as precursors in the cytosol and imported into mitochondria by the general translocase TOM and the sorting machinery SAM. The SAM complex contains two proteins essential for cell viability, the channel-forming Sam50 and Sam35. We have identified the sorting signal of mitochondrial beta-barrel proteins that is universal in all eukaryotic kingdoms. The beta-signal initiates precursor insertion into a hydrophilic, proteinaceous membrane environment by forming a ternary complex with Sam35 and Sam50. Sam35 recognizes the beta-signal, inducing a major conductance increase of the Sam50 channel. Subsequent precursor release from SAM is coupled to integration into the lipid phase. We propose that a two-stage mechanism of signal-driven insertion into a membrane protein complex and subsequent integration into the lipid phase may represent a general mechanism for biogenesis of beta-barrel proteins.

Successive translocation into and out of the mitochondrial matrix: Targeting of proteins to the intermembrane space by a bipartite signal peptide

We investigated the import and sorting pathways of cytochrome b2 and cytochrome c1, which are functionally located in the intermembrane space of mitochondria. Both proteins are synthesized on cytoplasmic ribosomes as larger precursors and are processed in mitochondria in two steps upon import. The precursors are first translocated across both mitochondrial membranes via contact sites into the matrix. Processing by the matrix peptidase leads to intermediate-sized forms, which are subsequently redirected across the inner membrane. The second proteolytic processing occurs in the intermembrane space. We conclude that the hydrophobic stretches in the presequences of the intermediate-sized forms do not stop transfer across the inner membrane, but rather act as transport signals to direct export from the matrix into the intermembrane space.

A J-protein is an essential subunit of the presequence translocase–associated protein import motor of mitochondria

Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase–associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.

Internal targeting signal of the BCS1 protein: a novel mechanism of import into mitochondria.

The BCS1 protein is anchored in the mitochondrial inner membrane via a single transmembrane domain and has an N(out)‐C(in) topology. Unlike the majority of nuclear encoded mitochondrial preproteins, the BCS1 protein does not contain an N‐terminal targeting sequence. A positively charged segment of amino acids which is located immediately C‐terminal to the transmembrane domain acts as an internal targeting signal. In order to function, we postulate that this sequence co‐operates with the transmembrane domain to form a tight hairpin loop structure. This loop is translocated across the inner membrane via the MIM/mt‐Hsp70 machinery in a membrane potential‐dependent manner. This novel mechanism of import and sorting of the BCS1 protein is proposed to represent a more general mechanism used by a number of inner membrane proteins.

Membrane Protein Degradation by AAA Proteases in Mitochondria: Extraction of Substrates from Either Membrane Surface

Two AAA proteases, each with its catalytic site at the opposite membrane surface, mediate the ATP-dependent degradation of mitochondrial inner membrane proteins. We demonstrate here that a model substrate polypeptide containing hydrophilic domains at both sides of the membrane can be completely degraded by either of the AAA proteases, if solvent-exposed domains are in an unfolded state. A short protein tail protruding from the membrane surface is sufficient to allow the proteolytic attack of an AAA protease that facilitates domain unfolding at the opposite side. Our results provide a rationale for the membrane arrangement of AAA proteases in mitochondria and demonstrate that degradation of membrane proteins by AAA proteases involves an active extraction of transmembrane segments and transport of solvent-exposed domains across the membrane.