The mitochondrial genome encodes only a handful of the mitochondrial proteins; hundreds of mitochondrial proteins are encoded in the nucleus, synthesised on cytosolic polyribosomes and subsequently imported into the organelle. Most of the proteins targeted to the mitochondria are synthesised as precursors with an N-terminal extension called presequence or targeting sequence. Following synthesis in the cytosol, general molecular chaperones keep the precursor protein in a loosely folded and import competent conformation. More specific cytosolic factors might be involved in assisting protein transport to the correct organelle. At the surface of mitochondria, the precursor interacts with specific receptors and is subsequently translocated across the membranes through the translocases of the outer and inner membranes.
The aim of this thesis has been to illuminate key steps of protein import into plant mitochondria by studying protein sorting, translocation and the involvement of molecular chaperones in the import process.
Do there exist molecular chaperones in the plant cell, which affect mitochondrial protein import? We isolated a post-microsomal cytosolic fraction from Spinacia oleracea and showed the presence of a proteinaceous cytosolic factor with a capacity to stimulate protein import by enhancing the kinetics of translocation rather than the binding capacity. We investigated the occurrence and induction of two mitochondrial molecular chaperones, heat shock protein 70 (Hsp70) and Hsp60, in normal and heat-stressed spinach plants. We found that whereas Hsp60 was totally localised to the matrix, 50% of the Hsp70 was associated with the mitochondrial inner membrane. In stressed plants, the Hsp70 was induced 2 to 3-fold and the total amount of the induced Hsp70 was associated with the membrane.
We have shown a unique property of higher plant mitochondria: the precursor of the F1b subunit of the ATP synthase was covalently modified upon binding to mitochondria. The modification was catalysed by an enzyme of the mitochondrial outer membrane and occured at the N-terminal region of the cleavable presequence of the precursor. The modification was ATP and Ca2+ dependent. This is the first report of a covalent modification of a mitochondrial precursor. The unmodified precursor protein was favoured for import, implicating that the modification might be removed prior to translocation across the outer membrane.
We have studied the mechanism of protein translocation into plant mitochondria. Sulfhydryl group reagents affected different events of the protein import process, such as the binding and conformational status of the precursor, the receptor independent bypass import pathway and protein transport across the mitochondrial inner membrane. The effect of sulfhydryl group reagents was dependent on energisation state of mitochondria. We concluded that the redox and the conformational status of the sulfhydryl groups located on the outer side of the inner membrane translocase components were essential for protein import.
Stockholm: Stockholm University , 1999. , 63 p.