Presequences

While it has been known for many years that the N-terminal presequence is sufficient to promote mitochondrial targeting and assembly, the subsequent interaction of the precursor molecule with the outer mitochondrial membrane and the uptake of the protein is still an area of active research. There seems little doubt, however, that there are proteins on the outer mitochondrial membrane which are required for the import process. The function of these proteins is uncertain, but they may act as receptors with the subsequent transfer through the membrane at proteinous pores located at contact sites between the inner and outer membranes. Several proteins have been identified which seem to play an important role as either receptor proteins or part of the import channel (Pfanner et al., 1991). Again, not all proteins seem to depend on this mechanism. Cytochrome c, which is loosely associated with the outer aspect of the inner mitochondrial membrane, can cross... 

The nuclear-encoded proteins are inserted into both inner and outer mitochondrial membranes, the intermembrane space, and the matrix and there are several different mechanisms involved. As mentioned above there is no apparent requirement for a presequence on proteins which insert specifically into the mitochondrial outer membrane. For proteins destined for the inner mitochondrial membrane, a stop-transfer mechanism is proposed. Thus some information in the peptide must stop the complete transfer of the protein into the mitochondrial matrix, enabling the protein to remain in the inner mitochondrial membrane. For some proteins in the intermembrane space (for example the Rieske iron-sulphur protein associated with the outer face of complex III), a particularly complicated import pathway... 

The Rieske protein in mitochondrial bci complexes is assembled when the protein is incorporated into the complex. The Rieske protein is encoded in the nucleus and synthesized in the cytosol with a mitochondrial targeting presequence, which is required to direct the apoprotein to the mitochondrial matrix. The C-terminus is then targeted back to the outside of the inner mitochondrial membrane where the Rieske cluster is assembled. In addition, the presequence is removed and the protein is processed to its mature size after the protein is inserted into the bci complex. In mammals, the presequence is cleaved in a single step by the core proteins 1 and 2, which are related to the general mitochondrial matrix processing protease (MPP) a and (3 subunits the bovine heart presequence is retained as a 8.0 kDa subunit of the complex (42, 107). In Saccharomyces cerevis-iae, processing occurs in two steps Initially, the yeast MPP removes 22 amino acid residues to convert the precursor to the intermediate form, and then the mitochondrial intermediate protease (MIP) removes 8 residues after the intermediate form is in the bci complex (47). Cleavage by MIP is independent of the assembly of the Rieske cluster Conversion of the intermediate to the mature form was observed in a yeast mutant that did not assemble any Rieske cluster (35). However, in most mutants where the assembly of the Rieske cluster is prevented, the amount of Rieske protein is drastically reduced, most likely because of instability (35, 44). 

Matrix proteins must pass from cytosolic polyribosomes through the outer and inner mitochondrial membranes to reach their destination. Passage through the two membranes is called translocation. They have an amino terminal leader sequence (presequence),... 

The above describes the major pathway of proteins destined for the mitochondrial matrix. However, certain proteins insert into the outer mitochoiidrial membrane facilitated by the TOM complex. Others stop in the intermembrane space, and some insert into the inner membrane. Yet others proceed into the matrix and then return to the inner membrane or intermembrane space. A number of proteins contain two signaling sequences—one to enter the mitochondrial matrix and the other to mediate subsequent relocation (eg, into the inner membrane). Certain mitochondrial proteins do not contain presequences (eg, cytochrome Cy which locates in the inter membrane space), and others contain internal presequences. Overall, proteins employ a variety of mechanisms and routes to attain their final destinations in mitochondria. 

The sequence features of presequences are well known typically they are from 20 to 80 residues long preferably contain basic residues, serine,... 

Roise, D. (1997). Recognition and binding of mitochondrial presequences during the import of proteins into mitochondria. / Bioenerg. Biomembr. 29, 19-27. 

Larsen et al. reported the enzymatic cleavage of a desB30 insuhn B-chain from a presequence (Lys(Boc))6. This spacer shifts the conformation of the growing peptide chain from a y9-structure to a random coil conformation and reduces pep-tide-chain aggregation, which otherwise causes serious synthetic problems. Nova-syn KA [18] was employed as a solid support, but unfortunately, no information about the enzyme used was reported [19]. 

This a-helix is amphipathic, containing patches of positively charged and hydrophobic amino acids, respectively, on opposite surfaces of the theoretical cylinder. The presequence is usually processed by the mitochondrial processing peptidase (MPP) and the mature protein is sorted to either the matrix, or to the inner membrane if it bears a hydrophobic stop-transfer sequence. Some mitochondrial proteins, mostly destined to the membranes, do not have cleavable N-terminal presequences but have internal targeting signals that are not well characterized (Pfanner and Geissler 2001). 

Fig-i Mitochondrial protein import machinery as defined in S. cerevisiae. TOM translo-case of the outer mitochondrial membrane SAM sorting and assembly machinery TIM translocase of the inner mitochondrial membrane MIA mitochondrial IMS import and assembly machine PAM presequence translocase associated motor IMP inner membrane protease MPP mitochondrial processing peptidase. The numbers on the individual Tom, Sam, Tim or Pam components represent their approximate molecular masses in kDa. See text for mechanistic details. Adopted from Dolezal et al. 2006... 

A hydrogenosomal N-terminal cleavable presequence was first noted in . vaginalis ferredoxin, a matrix protein, when purified endogenous ferredoxin was found to lack eight amino acid residues at the N-terminus, relative to the... 

Table 1 Presequences of precursors to hydrogenosomal and mitosomal proteinsa... 

The I vaginalis hydrogenosomal presequences are generally short, ranging from 5 to 14 amino acid residues for those that have been proven experimentally, and up to 17 residues for the predicted presequences (Table 1). The presequences are enriched in the amino acid residues Ser (20%), Leu (14%), Arg (11%), Ala (8%), Phe (7%), Val (6%), Thr (6%) and Asn (5%). The other amino acids are significantly under-represented. Incidentally, or accidentally, the three amino acids most commonly found in these presequences, Ser, Leu and Arg, are the ones that are each encoded by six codons. This may have been relevant in the evolution of these presequences. The mitochondrial matrix N-terminal presequences are enriched in Arg (14%), Leu (12%), Ser (11%) and Ala (14%). On the other hand, chloroplast leader peptides have a different amino acid composition with 19% Ser and 9% Thr (von Heijne et al. 1989). Markedly under-represented in hydrogenosomal presequences are the acidic residues, as in the case of both mitochondrial and plastidic presequences (von Heijne et al. 1989). 

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