Wolfgang Voos

Global Analysis of the Mitochondrial N-Proteome Identifies a Processing Peptidase Critical for Protein Stability

Many mitochondrial proteins are synthesized with N-terminal presequences that are removed by specific peptidases. The N-termini of the mature proteins and thus peptidase cleavage sites have only been determined for a small fraction of mitochondrial proteins and yielded a controversial situation for the cleavage site specificity of the major mitochondrial processing peptidase (MPP). We report a global analysis of the N-proteome of yeast mitochondria, revealing the N-termini of 615 different proteins. Significantly more proteins than predicted contained cleavable presequences. We identified the intermediate cleaving peptidase Icp55, which removes an amino acid from a characteristic set of MPP-generated N-termini, solving the controversial situation of MPP specificity and suggesting that Icp55 converts instable intermediates into stable proteins. Our results suggest that Icp55 is critical for stabilization of the mitochondrial proteome and illustrate how the N-proteome can serve as rich source for a systematic analysis of mitochondrial protein targeting, cleavage and turnover.

Molecular chaperones as essential mediators of mitochondrial biogenesis

Chaperone proteins have been initially identified by their ability to confer cellular resistance to various stress conditions. However, molecular chaperones participate also in many constitutive cellular processes. Mitochondria contain several members of the major chaperone families that have important functions in maintaining mitochondrial function. The major Hsp70 of the mitochondrial matrix (mtHsp70) is essential for the translocation of cytosolic precursor proteins across the two mitochondrial membranes. MtHsp70 interacts with the preprotein in transit in an ATP-dependent reaction as it emerges from the translocation channel of the inner membrane. Together with two essential partner proteins, Tim44 and Mge1, mtHsp70 forms a membrane-associated import motor complex responsible for vectorial polypeptide movement and unfolding of preprotein domains. Folding of newly imported proteins in the matrix is assisted by the soluble chaperone system formed by mtHsp70 and its partner protein Mdj1. For certain substrate proteins, the protected folding environment that is offered by the large oligomeric Hsp60 complex facilitates further folding reactions. The mitochondrial Hsp70 Ssq1 is involved in the assembly of mitochondrial Fe/S clusters together with another member of the DnaJ family, Jac1. Chaperones of the Clp/Hsp100 family mediate the prevention of aggregation under stress conditions and eventually the degradation of mitochondrial proteins. Together, the chaperones of the mitochondrial matrix form a complex interdependent chaperone network that is essential for most reactions of mitochondrial protein biogenesis.

Parkinson phenotype in aged PINK1-deficient mice is accompanied by progressive mitochondrial dysfunction in absence of neurodegeneration

Background Parkinsons disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD. Methodology/Principal Findings Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of α-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons. Conclusion Thus, aging Pink1−/− mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.

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.

Pam16 has an essential role in the mitochondrial protein import motor.

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70–Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.

MECHANISMS OF PROTEIN TRANSLOCATION INTO MITOCHONDRIA

Mitochondrial biogenesis utilizes a complex proteinaceous machinery for the import of cytosolically synthesized preproteins. At least three large multisubunit protein complexes, one in the outer membrane and two in the inner membrane, have been identified. These translocase complexes cooperate with soluble proteins from the cytosol, the intermembrane space and the matrix. The translocation of presequence-containing preproteins through the outer membrane channel includes successive electrostatic interactions of the charged mitochondrial targeting sequence with a chain of import components. Translocation across the inner mitochondrial membrane utilizes the energy of the proton motive force of the inner membrane and the hydrolysis of ATP. The matrix chaperone system of the mitochondrial heat shock protein 70 forms an ATP-dependent import motor by interaction with the polypeptide chain in transit and components of the inner membrane translocase. The precursors of integral inner membrane proteins of the metabolite carrier family interact with newly identified import components of the intermembrane space and are inserted into the inner membrane by a second translocase complex. A comparison of the full set of import components between the yeast Sacccharomyces cerevisiae and the nematode Caenorhabditis elegans demonstrates an evolutionary conservation of most components of the mitochondrial import machinery with a possible greater divergence for the import pathway of the inner membrane carrier proteins.

Protein unfolding by mitochondria. The Hsp70 import motor.

Protein unfolding is a key step in the import of some proteins into mitochondria and chloroplasts and in the degradation of regulatory proteins by ATP‐dependent proteases. In contrast to protein folding, the reverse process has remained largely uninvestigated until now. This review discusses recent discoveries on the mechanism of protein unfolding during translocation into mitochondria. The mitochondria can actively unfold preproteins by unraveling them from the N‐terminus. The central component of the mitochondrial import motor, the matrix heat shock protein 70, functions by both pulling and holding the preproteins.

Tim50 Maintains the Permeability Barrier of the Mitochondrial Inner Membrane

Transport of metabolites across the mitochondrial inner membrane is highly selective, thereby maintaining the electrochemical proton gradient that functions as the main driving force for cellular adenosine triphosphate synthesis. Mitochondria import many preproteins via the presequence translocase of the inner membrane. However, the reconstituted Tim23 protein constitutes a pore remaining mainly in its open form, a state that would be deleterious in organello. We found that the intermembrane space domain of Tim50 induced the Tim23 channel to close. Presequences overcame this effect and activated the channel for translocation. Thus, the hydrophilic cis domain of Tim50 maintains the permeability barrier of mitochondria by closing the translocation pore in a presequence-regulated manner.

Chapter 11 Assaying protein import into mitochondria

Publisher Summary Most proteins found in the mitochondria originate in the cytosol and therefore must be imported into this organelle following their synthesis. Most of the translocase components of the outer and inner mitochondrial membranes (TOM and TIM, respectively) have been identified in Saccharomyces cerevisiae and Neurospora crassa . A preprotein is directed to the mitochondria by virtue of its targeting signal, which is most often situated at the N terminus. Preproteins are first bound to receptors of the mitochondrial membrane—Tom70, Tom22, and Tom20—before being translocated across the outer membrane channel, which is formed by Tom40. Many preproteins destined to reside in the outer membrane transverse laterally out of the channel upon contact of their hydrophobic anchors with the TOM machinery. Further, another group of inner membrane proteins contain internal targeting signals that direct them from the inner face of the TOM machinery to a different TIM complex (the TIM22 complex) via their association with small intermembrane space TIM proteins. The characterization of these different import pathways has been achieved mainly through the use of mitochondrial in vitro import studies. In such cases, isolated mitochondria are incubated with in vitro -translated 35 S-labeled preproteins under varying conditions, and their import characteristics and requirements are analyzed. This chapter describes various methods employed to characterize both the mitochondrial import of preproteins and the protein import machinery in vitro .

Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins.

The mitochondrial heat shock protein Hsp70 is essential for import of nuclear‐encoded proteins, involved in both unfolding and membrane translocation of preproteins. mtHsp70 interacts reversibly with Tim44 of the mitochondrial inner membrane, yet the role of this interaction is unknown. We analysed this role by using two yeast mutants of mtHsp70 that differentially influenced its interaction with Tim44. One mutant mtHsp70 (Ssc1–2p) efficiently bound preproteins, but did not show a detectable complex formation with Tim44; the mitochondria imported loosely folded preproteins with wild‐type kinetics, yet were impaired in unfolding of preproteins. The other mutant Hsp70 (Ssc1–3p’) bound both Tim44 and preproteins, but the mitochondria did not import folded polypeptides and were impaired in import of unfolded preproteins; Ssc1–3p′ was defective in its ATPase domain and did not undergo a nucleotide‐dependent conformational change, resulting in permanent binding to Tim44. The following conclusions are suggested. (i) The import of loosely folded polypeptides (translocase function of mtHsp70) does not depend on formation of a detectable Hsp70‐Tim44 complex. Two explanations are possible: a trapping mechanism by soluble mtHsp70, or a weak/very transient interaction of Ssc1–2p with Tim44 that leads to a weak force generation sufficient for import of loosely folded, but not folded, polypeptides. (ii) Import of folded preproteins (unfoldase function of mtHsp70) involves a reversible nucleotide‐dependent interaction of mtHsp70 with Tim44, including a conformational change in mtHsp70. This is consistent with a model that the dynamic interaction of mtHsp70 with Tim44 generates a pulling force on preproteins which supports unfolding during translocation.