Import machinery

Too little maintenance results in unexpected failures and consequential major losses of production and/or customers (Figure 7-1). This impractical approach is termed reactive strategy and should be avoided on all important machinery. Optimum maintenance strategy balances reasonable costs with maximum possible availability and reliability. The two main maintenance strategies employed by companies today are labelled predictive strategy and preventive strategy. These are part of a balanced approach as shown in Figure 7-2. 

Sequence of amino acids that determine the transport of proteins into the nucleus. Although there is no clear consensus, nuclear localization signals tend to be rich in positively charged residues, which allow interaction with proteins from the nuclear import machinery (i.e., importins). 

Pfanner, N., Rossow, J., van der Klei, I.J., Neupert W. (1992). A dynamic model of the mitochondrial protein import machinery. Cell 68,999-1002. 

Defects of nuclear DNA also cause mitochondrial diseases. As mentioned above, the vast majority of mitochondrial proteins are encoded by nDNA, synthesized in the cytoplasm and imported into the mitochondria through a complex series of steps. Diseases can be due to mutations in genes encoding respiratory chain subunits, ancillary proteins controlling the proper assembly of the respiratory chain complexes, proteins controlling the importation machinery, or proteins controlling the lipid composition of the inner membrane. All these disorders will be transmitted by mendelian inheritance. From a biochemical point of view, all areas of mitochondrial metabolism can be affected (see below). 

Kessler F, Blobel G, Patel A, Schnell DJ. Identification of the two GTP-binding proteins in the chloroplast import machinery. Science 1994 266 1035-1039. 

SchnellDJ, KesslerF, BlobelG. Isolation of components ofthe chloroplast protein import machinery. Science 1994 266 1007-1012. 

Eckert, J. H. and Johnsson, N. PexlOp links the ubiquitin conjugating enzyme Pex4p to the protein import machinery of the peroxisome. J. Cell Sci. 2003, 336, 3623-34. 

Based on the intrinsic mitochondriotropism of dequalinium and its unique self-assembly behavior, we have developed a strategy for direct mitochondrial transfection (47-49), which involves the transport of a DNA-mitochondrial leader sequence (MLS) peptide conjugate to mitochondria using DQAsomes, the liberation of this conjugate from the cationic vector upon contact with the mitochondrial outer membrane followed by DNA uptake via the mitochondrial protein import machinery. We have demonstrated that DQAsomes fulfill all essential prerequisites for a mitochondria-specific DNA delivery system they bind and condense pDNA (24), protect it from... 

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... 

It has been hypothesized that the process of inventing a protein import machine for mitochondria would have been so intricate and critical that it is unlikely to have occurred more than once (CavaUer-Smith 1987). By extension, similarities found between mitosomal and hydrogenosomal and mitochondrial protein import have been presented as strong support that all these organelles use common components for import, and are therefore one and the same. These observations have prompted a number of experimental and bioinformatics studies to shed light on the constitution and evolution of the protein import machineries of hydrogenosomes and mitosomes. 

Two major protein import machineries have been characterized to date in the mitochondrial outer membrane the TOM and the SAM complexes (Fig. 1). 

Dekker PJ, Keil P, Rassow J, Maarse AC, Pfanner N, Meijer M (1993) Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane. FEBS Lett 330 66-70... 

Pfanner N, Geissler A (2001) Versatility of the mitochondrial protein import machinery. Nat Rev Mol Cell Biol 2 339-349... 

Plumper E, Bradley PJ, Johnson PJ (2000) Competition and protease sensitivity assays provide evidence for the existence of a hydrogenosomal protein import machinery in Trichomonas vaginalis. Mol Biochem Parasitol 106 11-20 Pool MR (2005) Signal recognition particles in chloroplasts, bacteria, yeast and mammals (review). Mol Membr Biol 22 3-15... 

A characterization of the translocase complexes responsible for mitosomal protein import in Giardia is under way. An iterative BLAST search of the G. intestinalis genome was employed for the identification of the import machinery components. The only candidate to have been found so far is GiPAMIS, a mitosomal protein homologous to mitochondrial presequence translocase-associated motor 18 (Dolezal et al. 2005). 

Wiedemann N, Frazier AE, Pfanner N (2004) The protein import machinery of mitochondria. J Biol Chem 279 14473-14476... 

Figure 12.5 Nuclear import in permeabilized cells. HeLa cells were grown on coverslips and permeabilized with digitonin as described in Wilson et al., 1999. Fluorescein-PNA-labeled plasmids (containing the SV40 enhancer, 4.2 kb) or rhodamine-labeled BSA-NLS peptide conjugates were incubated with the cells for four hours at which time they were viewed by fluorescence microscopy. With no additions, neither DNA nor protein was imported, but in the presence of nuclear and cytoplasmic extracts both substrates localized to the nuclei. While plasmids containing the SV40 enhancer were taken up by the nuclei, those lacking the sequence were excluded. The remaining panels demonstrate the need for both the import machinery (importins and Ran) and a source of adapter proteins (nuclear extract) for plasmid nuclear entry, but not for protein nuclear localization.

Figure 12.8 Model for general and cell-specific plasmid nuclear import. (A) SV40 enhancer-mediated nuclear import. Because the transcription factors bound by this DNA sequence are ubiquitously expressed, SV40 DNA localizes to the nuclei of all cell types (see Table 12.1). (B) Smooth muscle-specific plasmid nuclear import. Smooth muscle-specific transcription factors, including SRF among others, can bind to their target sites within the SMGA promoter carried on a plasmid and serve to transport the DNA to the nucleus via interactions with the NLS-mediated protein import machinery. Since these factors are not expressed in other cell types, no nuclear import will occur in non-smooth muscle cells.

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