Import of Yeast Mitochondrial Transcription Factor (Mtf1p) via a Nonconventional Pathway

Abstract

The yeast mitochondrial (mt) transcription factor Mtf1p is imported into the mitochondria from the cytoplasm without a conventional mt-targeting presequence. To understand its import the mt translocation of wild type and mutant Mtf1p constructs was investigated in vitro under various assay conditions. We report here that Mtf1p, unlike most mt matrix proteins hitherto studied, is translocated into the mitochondria independent of membrane potential, ATP hydrolysis, and membrane receptor. This unusual import of Mtf1p was also observed on ice (3°C). Sub-mitochondrial fractionation demonstrated that Mtf1p was translocated in vitro to one or more of the same mt sites as the endogenous protein that includes the matrix. To identify the mt-targeting sequence of Mtf1p, various N-terminal, C-terminal, or internally deleted Mtf1p derivatives were generated. The full-length and C-terminal deletions but not the N-terminal truncated Mtf1p were imported into mitochondria, indicating the importance of its N-terminal sequence for mt targeting. However, the internal deletion of Mtf1p revealed that the first 150-amino acid N-terminal sequence alone was not sufficient for mt targeting of Mtf1p, suggesting that an extended rather than a short N-terminal sequence is required for import. We favor a model in which Mtf1p adopts an import-competent conformation during translation. Consistent with this model are three findings: most of the protein sequence appears to be required for optimal import, urea denaturation eliminates its import competence, and the import-competent form of the protein is more resistant to tryptic hydrolysis than is the denatured protein. This represents a novel mechanism for mitochondrial protein import.Mitochondria and chloroplasts are unique among eukaryotic organelles in possessing their own genomes. These extra-chromosomal DNAs are expressed by the organelle-specific transcription and translational machinery (1-5). The yeast mt 1 genome encodes seven protein subunits of the energy-transducing enzyme complexes imbedded in the inner membrane, one ribosomal protein (i.e. Var1) of the small mitoribosomal subunit, two rRNAs (i.e. 21 S and 15 S rRNAs for the large and small subunits, respectively), a complete set of tRNAs, and an

Transcription Factor-dependent DNA Bending Governs Promoter Recognition by the Mitochondrial RNA Polymerase

“…Similarly, introduction of a single bp change of G:C to C:G at the Ϫ2 position in the promoter made the -2C:G-mt20 transcriptionally inactive (Fig. 1B, lane 2), which is consistent with the importance of the Ϫ2 bp reported in previous studies (28) and further demonstrating the high promoter specificity of Rpo41-Mtf1. Rpo41 alone showed no specific RNA products with any of the DNA fragments, which is also consistent with previous transcriptional studies (13,17).…”

“…The dramatic effect of a single base pair change indicates a cooperative mechanism of promoter selection whereby all base-specific interactions are required to sharply bend and melt the DNA. Our results indicate that transcription factor-enhanced DNA bending and melting rather than protein-DNA binding plays an important role in transcription efficiency and loss of an induced conformational change is responsible for the reduction or inactivation of transcription efficiency documented for sequence-mutated promoter variants (28,29,36,37).…”

“…It is interesting that a single bp transversion from G:C to C:G at position -2 abolishes DNA bending and melting, which is required for the formation of competent pre-initiation open complex. This explains the important role of -2 G:C bp of the promoter in initiating transcription (28).…”

The Ustilago maydis a2 Mating-Type Locus Genes lga2 and rga2 Compromise Pathogenicity in the Absence of the Mitochondrial p32 Family Protein Mrb1

“…In line with the basic nature of Rga2, many of the described interaction partners of p32 family proteins are characterized by basic regions (T. brucei RBP16, human ASF/SF2, and Epstein-Barr nuclear antigen-1), suggesting the participation of electrostatic interactions (Hayman et al, 2001 and references therein). The fact that Rga2 localizes to mitochondria in the absence of an N-terminal targeting sequence implicates the existence of an alternative targeting mechanism for which there is precedence (Biswas and Getz, 2002;Stan et al, 2003). At present we have no data concerning functional domains for the mitochondrial localization of Rga2, but its presence in mitochondria does not require Mrb1.…”

Protein Import into Hydrogenosomes ofTrichomonas vaginalisInvolves both N-Terminal and Internal Targeting Signals: a Case Study of Thioredoxin Reductases

“…Hydrogenosomes and mitochondria are homologous organelles which descend from the same eubacterial endosymbiont (32,47) (29). Though the first three examples represent mitochondrial carrier family proteins which are expected to have multiple internal signals, nonconventional import pathways were also reported for matrix proteins, specifically for the nuclearly encoded mitochondrial transcription factor Mtf1p (2). Its translocation appears to be independent of a cleavable Nterminal presequence, and Mtf1p seems to be capable of using alternative import signals present in different regions of the protein (3).…”