Requirement of different mitochondrial targeting sequences of the yeast mitochondrial transcription factor Mtf1p when synthesized in alternative translation systems

Abstract

Mitochondrial (mt) translocation of the nuclearly encoded mt transcription factor Mtf1p appears to occur independent of a cleavable presequence, mt receptor, mt membrane potential or ATP [Biswas and Getz (2002) J. Biol. Chem. 277, 45704-45714]. To understand further the import strategy of Mtf1p, we investigated the import of the wild-type and N-terminal-truncated Mtf1p mutants synthesized in two different in vitro translation systems. These Mtf1p derivatives were generated either in the RRL (rabbit reticulocyte lysate) or in the WGE (wheat germ extract) translation system. Under the in vitro import conditions, the RRL-synthesized full-length Mtf1p but not the N-terminal-truncated Mtf1p product was efficiently imported into mitochondria, suggesting that the N-terminal sequence is important for its import. On the other hand, when these Mtf1p products were generated in the WGE system, surprisingly, the N-terminal-truncated products, but not the full-length protein, were effectively translocated into mitochondria. Despite these differences between the translation systems, in both cases, import occurs at a low temperature and has no requirement for a trypsin-sensitive mt receptor, mt membrane potential or ATP hydrolysis. Together, these observations suggest that, in the presence of certain cytoplasmic factors (derived from either RRL or WGE), Mtf1p is capable of using alternative import signals present in different regions of the protein. This appears to be the first example of usage of different targeting sequences for the transport of a single mt protein into the mt matrix.

Trypanosoma brucei: Differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

“…Mitochondrial transcription factor Mtf1P in yeast does not depend on either Δψ or matrix ATP for its import (Biswas and Getz 2002). Furthermore, this protein depends on the N-terminal targeting signal for its import, when synthesized on rabbit reticulicyte translation system, but does not depend on this signal while the precursor protein was synthesized in the wheat germ extract, suggesting that the same protein is capable of utilizing targeting signals located at different regions of the protein depending on the cytosolic factor(s) present in the specific translation system (Biswas and Getz, 2004). In addition, T. brucei cyt c1 also known to be imported in the procyclic mitochondria in the presence of Δψ inhibitors, whereas, in other system import of cyt c1 is strictly dependent on Δψ (Priest and Hajduk, 2003).…”

A novel function of the mitochondrial transcription factor Mtf1 in fission yeast; Mtf1 regulates the nuclear transcription of srk1

“…Proteins can be imported co-translationally or post-translationally ( 41–44 ). The Mtf1 from S. cerevisiae has been shown to employ a unique mechanism for mitochondrial transport that does not require an N-terminal presequence, or an outer membrane receptor, is independent of the energy state of the mitochondria and can occur at low temperatures ( 45–47 ). The mechanisms of mitochondrial transport in S. pombe are not well understood.…”

Functional Definition of Outer Membrane Proteins Involved in Preprotein Import into Mitochondria

“…However, the following should be noted: (1) the F A d precursor is as efficiently imported into mitochondria from a wheat germ lysate compared with a rabbit reticulocyte lysate (Dessi et al, 1996;Tanudji et al, 2001), and (2) a study analyzing the inhibitory effect of wheat germ on import indicated that it is due to the folding status of the mature part of the protein (Dessi et al, 2003). Other precursor proteins can also be imported into mitochondria from a wheat germ lysate (Biswas and Getz, 2004). Thus, it appears that some formulations of the wheat germ translation lysate contain factors that inhibit import, and, notably, it has been reported that other factors, such as mitochondrial import stimulating factor, can relieve this inhibition (Hachiya et al, 1993).…”