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In both mitochondria and chloroplasts, protein import requires energy and occurs at points where the outer and inner organelle membranes are in close contact. Because mitochondria and chloroplasts contain multiple membranes and membrane-limited spaces, sorting of many proteins to their correct location often requires the sequential action of two targeting sequences and two membrane-bound translocation systems: one to direct the protein into the organelle, and the other to direct it into the correct organellar compartment or membrane. As we will see, the mechanisms for sorting various proteins to mitochondria and chloroplasts are related to some of the mechanisms discussed previously.

Amphipathic N-Terminal Signal Sequences Direct Proteins to the Mitochondrial Matrix

All proteins that travel from the cytosol to the same mito-chondrial destination have targeting signals that share common motifs, although the signal sequences are generally not identical. Thus the receptors that recognize such signals are able to bind to a number of different but related sequences. The most extensively studied sequences for localizing proteins to mitochondria are the matrix-targeting sequences. These sequences, located at the N-terminus, are usually 20-50 amino acids in length. They are rich in hydrophobic amino acids, positively charged basic amino acids (arginine and lysine), and hydroxylated ones (serine and threonine), but tend to lack negatively charged acidic residues (aspartate and glutamate).

Mitochondrial matrix-targeting sequences are thought to assume an a-helical conformation in which positively charged amino acids predominate on one side of the helix and hydrophobic amino acids predominate on the other side; thus these sequences are amphipathic. Mutations that disrupt this amphipathic character usually disrupt targeting to the matrix, although many other amino acid substitutions do not. These findings indicate that the amphipathicity of matrix-targeting sequences is critical to their function.

The cell-free assay outlined in Figure 16-25 has been widely used in studies on the import of mitochondrial precursor proteins. In this system, respiring (energized) mitochondria extracted from cells can incorporate mitochondrial precursor proteins carrying appropriate uptake-targeting sequences that have been separately synthesized in the absence of mitochondria. Successful incorporation of the precursor into the organelle can be assayed either by resistance to digestion by an exogenously added protease or, in most cases, by cleavage of the N-terminal targeting sequences by specific proteases. The uptake of completely synthesized mitochondrial precursor proteins by the organelle in this system contrasts with the cellfree cotranslational translocation of secretory proteins, which generally occurs only when microsomal membranes are present during synthesis (see Figure 16-4).

Mitochondrial Protein Import Requires Outer-Membrane Receptors and Translocons in Both Membranes

Figure 16-26 presents an overview of protein import from the cytosol into the mitochondrial matrix, the route into the mitochondrion followed by most imported proteins. We will discuss in detail each step in protein transport into the matrix

Uptake-

targeting sequence

Mitochondrial protein

Uptake-

targeting sequence

Mitochondrial protein

Add energized yeast mitochondria

Yeast mitochondrial proteins made by cytoplasmic ribosomes in a cell-free system i.

Add energized yeast mitochondria

Yeast mitochondrial proteins made by cytoplasmic ribosomes in a cell-free system

Protein taken up into mitochondria; uptake-targeting sequence removed and degraded

Trypsin

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