Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase

Abstract

Alignment of three fungal mtRNA polymerases revealed conserved amino acid sequences in an amino-terminal region of the Saccharomyces cerevisiae enzyme implicated previously as harboring an important functional domain. Phenotypic analysis of deletion and point mutations, in conjunction with a yeast two-hybrid assay, revealed that Nam1p, a protein involved in RNA processing and translation in mitochondria, binds specifically to this domain. The significance of this interaction in vivo was demonstrated by the fact that the temperature-sensitive phenotype of a deletion mutation (rpo41Delta2), which impinges on this amino-terminal domain, is suppressed by overproducing Nam1p. In addition, mutations in the amino-terminal domain result specifically in decreased steady-state levels of mature mitochondrial CYTB and COXI transcripts, which is a primary defect observed in NAM1 null mutant yeast strains. Finally, one point mutation (R129D) did not abolish Nam1p binding, yet displayed an obvious COX1/CYTB transcript defect. This mutation exhibited the most severe mitochondrial phenotype, suggesting that mutations in the amino-terminal domain can perturb other critical interactions, in addition to Nam1p binding, that contribute to the observed phenotypes. These results implicate the amino-terminal domain of mtRNA polymerases in coupling additional factors and activities involved in mitochondrial gene expression directly to the transcription machinery.

Fig. 1. A region of the sc-mtRNA polymerase amino-terminal extension is conserved in two other fungal species

A linear representation of sc-mtRNA polymerase (encoded by the RPO41 gene) is presented at the top of the figure. The carboxyl-terminal portion of the enzyme that is homologous to bacteriophage RNA polymerases is depicted as a gray box, the mitochondrial targeting sequence by a black box, and the amino-terminal extension by a white box. Expanded at the bottom is an alignment of the most conserved portion of the amino-terminal extensions from S. cerevisiae (S.c.), amino acids 110–205; N. crassa (N.c.), amino acids 122–219; and S. pombe (S.p.), amino acids 34–117. Amino acid residues that are identical are indicated by darker shading, and those that are similar are indicated by lighter shading. The residues in sc-mtRNA polymerase that were changed by site-directed mutagenesis are indicated by arrows, at the ends of the arrows the amino acid substitution is shown (as well as the RPO41 allele designations, which are boxed). Also indicated is the end point of two deletion mutations that were characterized previously (15). The rpo41Δ2 allele deletes amino acids 27–117 (end point labeled Δ2), and thus partially impinges on the conserved region. The rpo41Δ3 allele deleted amino acids 27–212 (end point labeled Δ3) and therefore completely removes the conserved region.

Fig. 2. Analysis of sc-mtRNA polymerase amino-terminal domain mutations

A, growth phenotypes. Shown to the left is the RPO41 allele expressed in each strain after plasmid shuffle in GS112. At the top of each column of panels, the growth medium (YPG or YPD) and growth temperature (30 or 37 °C) are indicated. For example, the strain that is expressing wild-type allele (RPO41) after plasmid shuffle is indicated. Serial 10-fold dilutions are plated from left to right within each panel and, in each row of panels, identical cultures were plated. B, western analysis of rpo41 point-mutant strains after plasmid shuffle. The blot was first probed with an anti-RPO41p antibody and the location of the 150-kDa, full-length mtRNA polymerase (Rpo41p) is indicated. The RPO41 null and point-mutant strains analyzed are indicated above each lane. The identical blot was stripped and probed again with an antibody against Yrb1p (yeast Ran-binding protein, a nuclear transport protein) to serve as a control for the amount of protein loaded in each lane.

Fig. 3. Nam1p interacts specifically with the amino-terminal extension of sc-mtRNA polymerase and suppresses a mutation in this region

A, β-galactosidase activity (dark color in lower panel) indicates a two-hybrid interaction between Nam1p and sc-mtRNA polymerase in the lacZ-reporter strain Y190, which that contains a plasmid (pAS1-RPO) encoding an intact amino-terminal domain. The same strain containing a plasmid (pAS-RPOΔ4) that is missing the amino-terminal domain, but contains other sc-mtRNA polymerase sequences, produces no β-galactosidase activity (upper panel, no dark color). B, overproduction of Nam1p suppresses the YPG growth defects of a rpo41Δ2 strain but not a rpo41Δ3 strain. Five strains that were streaked onto a YPG plate and grown at 36 °C are shown. The yeast strain GS124 contains a plasmid encoding the rpo41Δ2 allele as its only source of sc-mtRNA polymerase; the yeast strain GS125 contains a plasmid encoding the rpo41Δ3 allele as its only source of sc-mtRNA polymerase; and GS122 is the isogenic wild-type strain that has the wild-type RPO41 gene provided on a plasmid as its only source of sc-mtRNA polymerase (15). GS124 and GS125 transformed with an empty URA3 plasmid (YEp352) or with a URA3 plasmid that overexpresses Nam1p (pYES/GS-NAM1) are indicated.

Fig. 4. Northern analysis of mitochondrial transcripts from amino-terminal domain mutant yeast strains

Five micrograms of mtRNA was analyzed from the yeast strains indicated at the top of each lane. RNA was isolated from the RPO41 mutant (Δ2, Δ3, E119A/C121A, N152A/Y154A, and R129D) and the corresponding isogenic wild-type strain (RPO41) after growing in SD medium for five generations at 37 °C. RNA was isolated from the NAM1 null mutant strain (nam1) and its corresponding isogenic wild-type strain (NAM1) after growing in SD medium at 30 °C. The resulting RNA blot was successively hybridized with COB, COX1, and COX3 exon probes (as indicated to the left of the figure). The size of each transcript detected is indicated in kb to the right of the figure.

Fig. 5. The amino-terminal extensions of vertebrate mtRNA polymerases have a repetitive amino acid motif similar to that found in CRP1, a protein involved in RNA-processing and translation in chloroplasts

The human mtRNA polymerase is diagrammed at the top in the same manner as the yeast enzyme in Fig. 1. A linear representation of the Zea mays CRP1 protein is also presented. The region of amino acid sequence similarity that is common to vertebrate (human and Xenopus) mtRNA polymerases and CRP1 is depicted as a hatched box. Shown at the bottom is a ClustalW (30) alignment of two regions in CRP1 (CRP-box1 and CRP-box2) with the analogous region of the human and Xenopus mtRNA polymerases. Black-boxed letters denote amino acid identity, and gray-boxed letters indicate amino acid similarity. Recent evidence suggests that these regions of CRP1 are composed of a tandemly repeated, 35-amino acid domain called a PPR motif that appears to define a new family of proteins involved in RNA processing and translation in organelles (29). Based on a proposed consensus sequence for a PPR motif (29), it appears that the vertebrate mtRNA polymerases contain at least two PPR repeats in the amino-terminal extension (indicated at the bottom of the figure).