TY - JOUR
T1 - Protein import and processing reconstituted with isolated rat liver mitochondria and recombinant mitochondrial processing peptidase
AU - Cavadini, Patrizia
AU - Gakh, Oleksandr
AU - Isaya, Grazia
N1 - Funding Information:
We thank H. O'Neill for help in editing the manuscript, and F. Kalousek and J. Adamec for helpful discussions. Work in the authors' laboratory is supported by grants from the Muscular Dystrophy Association and the National Institute on Aging/NIH (AG15709). P.C. is supported by the Pierfranco and Luisa Mariani Foundation, Milan, Italy.
PY - 2002
Y1 - 2002
N2 - Most mitochondrial proteins are synthesized in the cytoplasm as larger precursors carrying N-terminal matrix-targeting presequences, and are subsequently transported to the mitochondria. The presequence mediates the interaction between the precursor polypeptide and components of the mitochondrial protein import machinery, a complex apparatus that is responsible for translocation of the precursor across the two mitochondrial membranes. Once the precursor has reached the mitochondrial matrix, the presequence is removed by the general mitochondrial processing peptidase (MPP). Some precursors undergo additional processing steps carried out by specialized processing peptidases. For most mitochondrial proteins, however, cleavage by MPP is the step that precedes folding and assembly into the native form. We describe methods to isolate import-competent mitochondria from rat liver and to perform import reactions with precursor proteins synthesized in vitro by coupled transcription-translation. We also describe methods to perform in vitro processing reactions of mitochondrial precursors by recombinant MPP and to identify the cleavage sites used by this enzyme.
AB - Most mitochondrial proteins are synthesized in the cytoplasm as larger precursors carrying N-terminal matrix-targeting presequences, and are subsequently transported to the mitochondria. The presequence mediates the interaction between the precursor polypeptide and components of the mitochondrial protein import machinery, a complex apparatus that is responsible for translocation of the precursor across the two mitochondrial membranes. Once the precursor has reached the mitochondrial matrix, the presequence is removed by the general mitochondrial processing peptidase (MPP). Some precursors undergo additional processing steps carried out by specialized processing peptidases. For most mitochondrial proteins, however, cleavage by MPP is the step that precedes folding and assembly into the native form. We describe methods to isolate import-competent mitochondria from rat liver and to perform import reactions with precursor proteins synthesized in vitro by coupled transcription-translation. We also describe methods to perform in vitro processing reactions of mitochondrial precursors by recombinant MPP and to identify the cleavage sites used by this enzyme.
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U2 - 10.1016/S1046-2023(02)00035-X
DO - 10.1016/S1046-2023(02)00035-X
M3 - Article
C2 - 12054920
AN - SCOPUS:0036024972
SN - 1046-2023
VL - 26
SP - 298
EP - 306
JO - Methods
JF - Methods
IS - 4
ER -