Membrane protein turnover by the m-AAA protease in mitochondria depends on the transmembrane domains of its subunits

AAA proteases are membrane-bound ATP-dependent proteases that are present in eubacteria, mitochondria and chloroplasts and that can degrade membrane proteins. Recent evidence suggests dislocation of membrane-embedded substrates for proteolysis to occur in a hydrophilic environment; however, next to nothing is known about the mechanism of this process. Here, we have analysed the role of the membrane-spanning domains of Yta10 and Yta12, which are conserved subunits of the hetero-oligomeric m-AAA protease in the mitochondria of Saccharomyces cerevisiae. We demonstrate that the m-AAA protease retains proteolytic activity after deletion of the transmembrane segments of either Yta10 or Yta12. Although the mutant m-AAA protease is still capable of processing cytochrome c peroxidase and degrading a peripheral membrane protein, proteolysis of integral membrane proteins is impaired. We therefore propose that transmembrane segments of m-AAA protease subunits have a direct role in the dislocation of membrane-embedded substrates.     

The m-AAA protease defective in hereditary spastic paraplegia controls ribosome assembly in mitochondria

AAA proteases comprise a conserved family of membrane bound ATP-dependent proteases that ensures the quality control of mitochondrial inner-membrane proteins. Inactivation of AAA proteases causes pleiotropic phenotypes in various organisms, including respiratory deficiencies, mitochondrial morphology defects, and axonal degeneration in hereditary spastic paraplegia (HSP). The molecular basis of these defects, however, remained unclear. Here, we describe a regulatory role of an AAA protease for mitochondrial protein synthesis in yeast. The mitochondrial ribosomal protein MrpL32 is processed by the m-AAA protease, allowing its association with preassembled ribosomal particles and completion of ribosome assembly in close proximity to the inner membrane. Maturation of MrpL32 and mitochondrial protein synthesis are also impaired in a HSP mouse model lacking the m-AAA protease subunit paraplegin, demonstrating functional conservation. Our findings therefore rationalize mitochondrial defects associated with m-AAA protease mutants in yeast and shed new light on the mechanism of axonal degeneration in HSP.     

OPA1 processing reconstituted in yeast depends on the subunit composition of the m-AAA protease in mitochondria

The morphology of mitochondria in mammalian cells is regulated by proteolytic cleavage of OPA1, a dynamin-like GTPase of the mitochondrial inner membrane. The mitochondrial rhomboid protease PARL, and paraplegin, a subunit of the ATP-dependent m-AAA protease, were proposed to be involved in this process. Here, we characterized individual OPA1 isoforms by mass spectrometry, and we reconstituted their processing in yeast to identify proteases involved in OPA1 cleavage. The yeast homologue of OPA1, Mgm1, was processed both by PARL and its yeast homologue Pcp1. Neither of these rhomboid proteases cleaved OPA1. The formation of small OPA1 isoforms was impaired in yeast cells lacking the m-AAA protease subunits Yta10 and Yta12 and was restored upon expression of murine or human m-AAA proteases. OPA1 processing depended on the subunit composition of mammalian m-AAA proteases. Homo-oligomeric m-AAA protease complexes composed of murine Afg3l1, Afg3l2, or human AFG3L2 subunits cleaved OPA1 with higher efficiency than paraplegin-containing m-AAA proteases. OPA1 processing proceeded normally in murine cell lines lacking paraplegin or PARL. Our results provide evidence for different substrate specificities of m-AAA proteases composed of different subunits and reveal a striking evolutionary switch of proteases involved in the proteolytic processing of dynamin-like GTPases in mitochondria.     

Oma1, a novel membrane-bound metallopeptidase in mitochondria with activities overlapping with the m-AAA protease

The integrity of the inner membrane of mitochondria is maintained by a membrane-embedded quality control system that ensures the removal of misfolded membrane proteins. Two ATP-dependent AAA proteases with catalytic sites at opposite membrane surfaces are key components of this proteolytic system. Here we describe the identification of a novel conserved metallopeptidase that exerts activities overlapping with the m-AAA protease and was therefore termed Oma1. Both peptidases are integral parts of the inner membrane and mediate the proteolytic breakdown of a misfolded derivative of the polytopic inner membrane protein Oxa1. The m-AAA protease cleaves off the matrix-exposed C-terminal domain of Oxa1 and processively degrades its transmembrane domain. In the absence of the m-AAA protease, proteolysis of Oxa1 is mediated in an ATP-independent manner by Oma1 and a yet unknown peptidase resulting in the accumulation of N- and C-terminal proteolytic fragments. Oma1 exposes its proteolytic center to the matrix side; however, mapping of Oma1 cleavage sites reveals clipping of Oxa1 in loop regions at both membrane surfaces. These results identify Oma1 as a novel component of the quality control system in the inner membrane of mitochondria. Proteins homologous to Oma1 are present in higher eukaryotic cells, eubacteria and archaebacteria, suggesting that Oma1 is the founding member of a conserved family of membrane-embedded metallopeptidases.     

Variable and tissue-specific subunit composition of mitochondrial m-AAA protease complexes linked to hereditary spastic paraplegia

The m-AAA protease, an ATP-dependent proteolytic complex in the mitochondrial inner membrane, controls protein quality and regulates ribosome assembly, thus exerting essential housekeeping functions within mitochondria. Mutations in the m-AAA protease subunit paraplegin cause axonal degeneration in hereditary spastic paraplegia (HSP), but the basis for the unexpected tissue specificity is not understood. Paraplegin assembles with homologous Afg3l2 subunits into hetero-oligomeric complexes which can substitute for yeast m-AAA proteases, demonstrating functional conservation. The function of a third paralogue, Afg3l1 expressed in mouse, is unknown. Here, we analyze the assembly of paraplegin into m-AAA complexes and monitor consequences of paraplegin deficiency in HSP fibroblasts and in a mouse model for HSP. Our findings reveal variability in the assembly of m-AAA proteases in mitochondria in different tissues. Homo-oligomeric Afg3l1 and Afg3l2 complexes and hetero-oligomeric assemblies of both proteins with paraplegin can be formed. Yeast complementation studies demonstrate the proteolytic activity of these assemblies. Paraplegin deficiency in HSP does not result in the loss of m-AAA protease activity in brain mitochondria. Rather, homo-oligomeric Afg3l2 complexes accumulate, and these complexes can substitute for housekeeping functions of paraplegin-containing m-AAA complexes. We therefore propose that the formation of m-AAA proteases with altered substrate specificities leads to axonal degeneration in HSP.     

AAA proteases with catalytic sites on opposite membrane surfaces comprise a proteolytic system for the ATP-dependent degradation of inner membrane proteins in mitochondria.

The mechanism of selective protein degradation of membrane proteins in mitochondria has been studied employing a model protein that is subject to rapid proteolysis within the inner membrane. Protein degradation was mediated by two different proteases: (i) the m-AAA protease, a protease complex consisting of multiple copies of the ATP-dependent metallopeptidases Yta1Op (Afg3p) and Yta12p (Rcalp); and (ii) by Ymelp (Ytallp) that also is embedded in the inner membrane. Ymelp, highly homologous to Yta1Op and Yta12p, forms a complex of approximately 850 kDa in the inner membrane and exerts ATP-dependent metallopeptidase activity. While the m-AAA protease exposes catalytic sites to the mitochondrial matrix, Ymelp is active in the intermembrane space. The Ymelp complex was therefore termed 'i-AAA protease'. Analysis of the proteolytic fragments indicated cleavage of the model polypeptide at the inner and outer membrane surface and within the membrane-spanning domain. Thus, two AAA proteases with their catalytic sites on opposite membrane surfaces constitute a novel proteolytic system for the degradation of membrane proteins in mitochondria.     

Impaired protein quality control system underlies mitochondrial dysfunction in skeletal muscle of streptozotocin-induced diabetic rats

Hyperglycaemia-related mitochondrial impairment is suggested as a contributor to skeletal muscle dysfunction. Aiming a better understanding of the molecular mechanisms that underlie mitochondrial dysfunction in type 1 diabetic skeletal muscle, the role of the protein quality control system in mitochondria functionality was studied in intermyofibrillar mitochondria that were isolated from gastrocnemius muscle of streptozotocin (STZ)-induced diabetic rats. Hyperglycaemic rats showed more mitochondria but with lower ATP production ability, which was related with increased carbonylated protein levels and lower mitochondrial proteolytic activity assessed by zymography. LC-MS/MS analysis of the zymogram bands with proteolytic activity allowed the identification of an AAA protease, Lon protease; the metalloproteases PreP, LAP-3 and MIP; and cathepsin D. The content and activity of the Lon protease was lower in the STZ animals, as well as the expression of the m-AAA protease paraplegin, evaluated by western blotting. Data indicated that in muscle from diabetic rats the mitochondrial protein quality control system was compromised, which was evidenced by the decreased activity of AAA proteases, and was accompanied by the accumulation of oxidatively modified proteins, thereby causing adverse effects on mitochondrial functionality.     

Translating m-AAA protease function in mitochondria to hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurodegenerative disorder that is characterized by progressive and cell-specific axonal degeneration. An autosomal recessive form of the disease is caused by mutations in paraplegin, which is a conserved subunit of the ubiquitous and ATP-dependent m-AAA protease in mitochondria. The m-AAA protease carries out protein quality control in the inner membrane of the mitochondria, suggesting a pathogenic role of misfolded proteins in HSP. A recent study demonstrates that the m-AAA protease regulates ribosome assembly and translation within mitochondria by controlling proteolytic maturation of a ribosomal subunit. Here, we will discuss implications of the dual role of the m-AAA protease in protein activation and degradation for mitochondrial dysfunction and axonal degeneration.     

Loss of m-AAA protease in mitochondria causes complex I deficiency and increased sensitivity to oxidative stress in hereditary spastic paraplegia

Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP). Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein. We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane. These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts. The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin. Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin-AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure. These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease.     

Prohibitins regulate membrane protein degradation by the m-AAA protease in mitochondria

Prohibitins comprise a protein family in eukaryotic cells with potential roles in senescence and tumor suppression. Phb1p and Phb2p, members of the prohibitin family in Saccharomyces cerevisiae, have been implicated in the regulation of the replicative life span of the cells and in the maintenance of mitochondrial morphology. The functional activities of these proteins, however, have not been elucidated. We demonstrate here that prohibitins regulate the turnover of membrane proteins by the m-AAA protease, a conserved ATP-dependent protease in the inner membrane of mitochondria. The m-AAA protease is composed of the homologous subunits Yta10p (Afg3p) and Yta12p (Rca1p). Deletion of PHB1 or PHB2 impairs growth of Deltayta10 or Deltayta12 cells but does not affect cell growth in the presence of the m-AAA protease. A prohibitin complex with a native molecular mass of approximately 2 MDa containing Phb1p and Phb2p forms a supercomplex with the m-AAA protease. Proteolysis of nonassembled inner membrane proteins by the m-AAA protease is accelerated in mitochondria lacking Phb1p or Phb2p, indicating a negative regulatory effect of prohibitins on m-AAA protease activity. These results functionally link members of two conserved protein families in eukaryotes to the degradation of membrane proteins in mitochondria.     

m-AAA protease-driven membrane dislocation allows intramembrane cleavage by rhomboid in mitochondria

Maturation of cytochrome c peroxidase (Ccp1) in mitochondria occurs by the subsequent action of two conserved proteases in the inner membrane: the m-AAA protease, an ATP-dependent protease degrading misfolded proteins and mediating protein processing, and the rhomboid protease Pcp1, an intramembrane cleaving peptidase. Neither the determinants preventing complete proteolysis of certain substrates by the m-AAA protease, nor the obligatory requirement of the m-AAA protease for rhomboid cleavage is currently understood. Here, we describe an intimate and unexpected functional interplay of both proteases. The m-AAA protease mediates the ATP-dependent membrane dislocation of Ccp1 independent of its proteolytic activity. It thereby ensures the correct positioning of Ccp1 within the membrane bilayer allowing intramembrane cleavage by rhomboid. Decreasing the hydrophobicity of the Ccp1 transmembrane segment facilitates its dislocation from the membrane and renders rhomboid cleavage m-AAA protease-independent. These findings reveal for the first time a non-proteolytic function of the m-AAA protease during mitochondrial biogenesis and rationalise the requirement of a preceding step for intramembrane cleavage by rhomboid.     

Dislocation by the m-AAA Protease Increases the Threshold Hydrophobicity for Retention of Transmembrane Helices in the Inner Membrane of Yeast Mitochondria

Sorting of mitochondrial inner membrane proteins is a complex process in which translocons and proteases function in a concerted way. Many inner membrane proteins insert into the membrane via the TIM23 translocon, and some are then further acted upon by the mitochondrial m-AAA protease, a molecular motor capable of dislocating proteins from the inner membrane. This raises the possibility that the threshold hydrophobicity for the retention of transmembrane segments in the inner membrane is different depending on whether they belong to membrane proteins that are m-AAA protease substrates or not. Here, using model transmembrane segments engineered into m-AAA protease-dependent proteins, we show that the threshold hydrophobicity for membrane retention measured in yeast cells in the absence of a functional m-AAA protease is markedly lower than that measured in its presence. Whether a given hydrophobic segment in a mitochondrial inner membrane protein will ultimately form a transmembrane helix may therefore depend on whether or not it will be exposed to the pulling force exerted by the m-AAA protease during biogenesis.     

Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease

FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.     

Import of proteins into mitochondria. Energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2 by isolated yeast mitochondria

Import of in vitro-synthesized cytochrome b2 (a soluble intermembrane space enzyme) was studied wih isolated yeast mitochondria. Import requires an electrochemical gradient across the inner membrane and is accompanied by cleavage of the precursor to the corresponding mature form. This conversion proceeds via an intermediate form of cytochrome b2, which can be detected as a transient species when mitochondria are incubated with the cytochrome b2 precursor for short times or at low temperatures. Conversion of the precursor to the intermediate form is energy-dependent and catalyzed by an o-phenanthroline-sensitive protease located in the soluble matrix. The intermediate is subsequently converted to mature cytochrome b2 in a reaction which is o-phenanthroline-insensitive and requires neither an energized inner membrane nor a soluble component of the intermembrane space. Whereas mature cytochrome b2 is soluble, the intermediate formed by isolated mitochondria is membrane-bound and exposed to the intermembrane space. The same intermediate is detected as a transient species during cytochrome b2 maturation in intact yeast cells (Reid, G. A., Yonetani, T., and Schatz, G (1982) J. Biol. Chem. 257, 13068-13074). The in vitro studies reported here suggest that a part of the cytochrome b2 precursor polypeptide chain is transported to the matrix where it is cleaved to a membrane-bound intermediate form by the same protease that processes polypeptides destined for the matrix space or for the inner membrane. In a second reaction, the cytochrome b2 intermediate is converted to mature cytochrome b2 which is released into the intermembrane space. The binding of heme is not necessary for converting the intermediate to the mature polypeptide.     

Loss of Drosophila i-AAA protease,dYME1L,causes abnormal mitochondria and apoptotic degeneration

Mitochondrial AAA (ATPases Associated with diverse cellular Activities) proteases i-AAA (intermembrane space-AAA) and m-AAA (matrix-AAA) are closely related and have major roles in inner membrane protein homeostasis. Mutations of m-AAA proteases are associated with neuromuscular disorders in humans. However, the role of i-AAA in metazoans is poorly understood. We generated a deletion affecting Drosophila i-AAA, dYME1L (dYME1L^del). Mutant flies exhibited premature aging, progressive locomotor deficiency and neurodegeneration that resemble some key features of m-AAA diseases. dYME1L^del flies displayed elevated mitochondrial unfolded protein stress and irregular cristae. Aged dYME1L^del flies had reduced complex I (NADH/ubiquinone oxidoreductase) activity, increased level of reactive oxygen species (ROS), severely disorganized mitochondrial membranes and increased apoptosis. Furthermore, inhibiting apoptosis by targeting dOmi (Drosophila Htra2/Omi) or DIAP1, or reducing ROS accumulation suppressed retinal degeneration. Our results suggest that i-AAA is essential for removing unfolded proteins and maintaining mitochondrial membrane architecture. Loss of i-AAA leads to the accumulation of oxidative damage and progressive deterioration of membrane integrity, which might contribute to apoptosis upon the release of proapoptotic molecules such as dOmi. Containing ROS level could be a potential strategy to manage mitochondrial AAA protease deficiency.Mitochondria dictate the survival and well being of the eukaryotic cells, but their unique genetic system and complex biophysical characteristics make for great challenges in maintaining organelle integrity and function.¹ One challenge is ensuring the proper assembly of the protein complexes carrying out mitochondrial functions. Most mitochondrial proteins are encoded by the nuclear genome and imported into the mitochondria after synthesis.² However, mitochondria also contain their own genome, which encodes the core components of the electron transport chain (ETC). The mitochondrion-encoded subunits of the ETC assemble on the inner mitochondrial membrane (IMM) with the nuclear-encoded ones. Unassembled polypeptides have to be removed to maintain the stoichiometry of the ETC complexes. Another challenge is the production of reactive oxygen species (ROS), the unavoidable by-products of electron transfer, which are generated mainly at complex I (NADH/ubiquinone oxidoreductase) and complex III (ubiquinol-cytochrome c oxidoreductase) in the ETC.³ Excessive ROS can damage proteins and impair mitochondrial functions.An elaborate system of chaperones and proteases has evolved to ensure mitochondrial proteostasis.⁴ The proteases are located in different submitochondrial compartments and carry out critical steps of mitochondrial biogenesis and turnover, including processing, assembly and degradation of mitochondrial proteins. Mitochondrial proteases of the AAA class (ATPases Associated with diverse cellular Activities) are the main regulators of proteostasis on the IMM,⁵ which houses many important complexes including those of the ETC. The catalytic domains of AAA proteases face either the matrix (mitochondrial m-AAA proteases) or the intermembrane space (IMS) (mitochondrial i-AAA protease).⁶ Despite their different topologies, mitochondrial m-AAA proteases and i-AAA protease share highly conserved protein structures and catalytic mechanism, and even an overlapping substrate specificity.⁷ Mutations in the mitochondrial m-AAA proteases are responsible for neurodegenerative disorders including hereditary spastic paraplegia (HSP), spinocerebellar ataxia (SCA28) and spastic ataxia neuropathy syndrome.^{8, 9, 10} However, the degenerative mechanisms remain elusive,¹¹ and the presence of multiple mitochondrial m-AAA proteases with redundant functions in eukaryotes complicates their analysis in animal models. By contrast, only one mitochondrial i-AAA protease has been identified in eukaryotic genomes. It coordinates mitochondrial fusion and fission,¹² and couples the mitochondrial dynamics to oxidative phosphorylation.¹³ Knocking down mitochondrial i-AAA protease in cultured cells perturbed mitochondrial morphology and sensitized cells to oxidative stress and apoptotic stimuli.^{14, 15, 16} However, the pathophysiological consequences of i-AAA loss of function at the animal level have been largely unknown. Yet, the absence of gene redundancy makes mitochondrial i-AAA protease particularly suitable for genetic studies exploring the function of mitochondrial AAA proteases in animal models.Drosophila melanogaster has been widely used to understand the biochemical processes underlying a variety of human diseases,¹⁷ including many mitochondrial disorders such as Parkinson''s disease.^{18, 19, 20} In these studies, some key phenotypes of mitochondrial diseases, such as impaired locomotor activities and neural and muscular degeneration, have been successfully recapitulated in Drosophila. Here we demonstrate that loss of mitochondrial i-AAA protease (dYME1L) in Drosophila melanogaster perturbs mitochondrial proteostasis, causes mitochondrial anomalies and triggers apoptotic degeneration in neurons and muscles.     

Presequence-dependent folding ensures MrpL32 processing by the m-AAA protease in mitochondria

m-AAA proteases exert dual functions in the mitochondrial inner membrane: they mediate the processing of specific regulatory proteins and ensure protein quality control degrading misfolded polypeptides to peptides. Loss of these activities leads to neuronal cell death in several neurodegenerative disorders. However, it is unclear how the m-AAA protease chooses between specific processing and complete degradation. A central and conserved function of the m-AAA protease is the processing of the ribosomal subunit MrpL32, which regulates ribosome biogenesis and the formation of respiratory complexes. Here, we demonstrate that the formation of a tightly folded domain harbouring a conserved CxxC-X(9)-CxxC sequence motif halts degradation initiated from the N-terminus and triggers the release of mature MrpL32. Oxidative stress impairs folding of MrpL32, resulting in its degradation by the m-AAA protease and decreased mitochondrial translation. Surprisingly, MrpL32 folding depends on its mitochondrial targeting sequence. Presequence-assisted folding of MrpL32 requires the complete import of the MrpL32 precursor before maturation occurs and therefore explains the need for post-translocational processing by the m-AAA protease rather than co-translocational cleavage by the general mitochondrial processing peptidase.     

MAP-1 and IAP-1, two novel AAA proteases with catalytic sites on opposite membrane surfaces in mitochondrial inner membrane of Neurospora crassa

Eukaryotic AAA proteases form a conserved family of membrane-embedded ATP-dependent proteases but have been analyzed functionally only in the yeast Saccharomyces cerevisiae. Here, we have identified two novel members of this protein family in the filamentous fungus Neurospora crassa, which were termed MAP-1 and IAP-1. Both proteins are localized to the inner membrane of mitochondria. They are part of two similar-sized high molecular mass complexes, but expose their catalytic sites to opposite membrane surfaces, namely, the intermembrane and the matrix space. Disruption of iap-1 by repeat-induced point mutation caused a slow growth phenotype at high temperature and stabilization of a misfolded inner membrane protein against degradation. IAP-1 could partially substitute for functions of its yeast homolog Yme1, demonstrating functional conservation. However, respiratory growth at 37 degrees C was not restored. Our results identify two components of the quality control system of the mitochondrial inner membrane in N. crassa and suggest that AAA proteases with catalytic sites exposed to opposite membrane surfaces are present in mitochondria of all eukaryotic cells.     

The first twelve amino acids (less than half of the pre-sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix.

Yeast cytochrome c oxidase subunit IV (an imported mitochondrial protein) is made as a larger precursor with a transient pre-sequence of 25 amino acids. If this pre-sequence is fused to the amino terminus of mouse dihydrofolate reductase (a cytosolic protein) the resulting fusion protein is imported into the matrix space, and cleaved to a smaller size, by isolated yeast mitochondria. We have now fused progressively shorter amino-terminal segments of the subunit IV pre-sequence to dihydrofolate reductase and tested each fusion protein for import into the matrix space and cleavage by the matrix-located processing protease. The first 12 amino acids of the subunit IV pre-sequence were sufficient to direct dihydrofolate reductase into the mitochondrial matrix, both in vitro and in vivo. However, import of the corresponding fusion protein into the matrix was no longer accompanied by proteolytic processing. Fusion proteins containing fewer than nine amino-terminal residues from the subunit IV pre-piece were not imported into isolated mitochondria. The information for transporting attached mouse dihydrofolate reductase into mitochondria is thus contained within the first 12 amino acids of the subunit IV pre-sequence.