Premium
Ribosome‐Nascent Protein Complex and Mitochondria Preparation to Study Ribosome‐Mitochondria Interactions
Author(s) -
MacKenzie James A,
Payne R. Mark
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb62-a
Subject(s) - ribosome , mitochondrion , microbiology and biotechnology , chemistry , biology , biochemistry , rna , gene
The human mitochondrial genome encodes only 13 proteins, meaning that mitochondrial function is dependent upon the targeting and import of nuclearly‐encoded proteins. The co‐translational model of mitochondrial protein import requires a specific interaction between ribosomes and mitochondria. Investigating this interaction in a cell‐free system requires ribosomes that stably carry a nascent protein and pure mitochondria. Although ribosome‐nascent protein complexes (RNCs) have been used in the past to study protein import into the endoplasmic reticulum (ER), there is a great deal of variation in the methods used to create them. We report here the conditions that need to be taken into account when creating RNCs. We found that translation temperature and time, as well as the amount of transcription reaction added to the translation reaction, are critical to maximally load ribosomes. When studying ribosome‐mitochondria interactions, the ribosome‐binding ER must be removed from mitochondria preparations. We show here for the first time that one method for removing ER contamination, EDTA/high salt treatment, does not disrupt mitochondrial function. In conclusion, the information presented here is essential for studying ribosome‐mitochondria interactions.