Identification of 12 New Yeast Mitochondrial Ribosomal Proteins Including 6 That Have No Prokaryotic Homologues

Abstract

Mitochondrial ribosomal proteins were studied best in yeast, where the small subunit was shown to contain about 35 proteins. Yet, genetic and biochemical studies identified only 14 proteins, half of which were predictable by sequence homology with prokaryotic ribosomal components of the small subunit. Using a recently described affinity purification technique and tagged versions of yeast Ykl155c and Mrp1, we isolated this mitochondrial ribosomal subunit and identified a total of 20 proteins, of which 12 are new. For a subset of the newly described ribosomal proteins, we showed that they are localized in mitochondria and are required for the respiratory competency of the yeast cells. This brings to 26 the total number of proteins described as components of the mitochondrial small ribosomal subunit. Remarkably, almost half of the previously and newly identified mitochondrial ribosomal components showed no similarity to any known ribosomal protein. Homologues could be found, however, in predicted protein sequences from Schizosaccharomyces pombe. In more distant species, putative homologues were detected for Ykl155c, which shares conserved motifs with uncharacterized proteins of higher eukaryotes including humans. Another newly identified ribosomal protein, Ygl129c, was previously shown to be a member of the DAP-3 family of mitochondrial apoptosis mediators.In yeast mitochondria, the majority of the characterized ribosomal proteins are essential for protein synthesis (for review see Ref. 1). Homologues of about 18 of the 21 prokaryotic small ribosomal subunit proteins can be identified by similarity searches in the yeast complete genomic sequence. However, the number of ribosomal proteins is larger in the small subunit of yeast mitochondria, estimated to be 33 or 36 (2, 3).Thus far, yeast mitochondrial ribosomal proteins have been identified either by the study of mutant strains with mitochondrial dysfunction (pet mutants; for review see Ref. 4) or by direct biochemical approaches involving isolation of mitochondria, purification of mitochondrial ribosomes, and protein separation followed by microsequencing (5). Mitochondrial ribosome purification has thus far been technically difficult, because the ribosomal proteins represent only 2-3% of the mitochondrial proteins (2). To date, these approaches have identified only a subset of the total number of mitochondrial ribosomal proteins. In yeast, the eukaryote in which most of the studies were performed, only 14 proteins of the small mitochondrial ribosomal subunit have been characterized experimentally (1, 6).We were originally interested in the study of the yeast YKL155C gene because it was found in a two-hybrid exhaustive screen to interact with the Prp11 splicing factor (7). To test whether this protein was associated with splicing factors under physiological conditions, we used the recently described tandem affinity purification technique (8) that allows the isolation of protein complexes by two successive affinity purification steps under mild conditions. Instea...

Trypanosoma brucei Mitochondrial Ribosomes

“…The other motifs indicate GTP binding, GTPase, methyltransferase, peptidyl-prolyl isomerase, helicase activities, and chaperone function. These proteins may have roles in ribosome maturation or assembly, RNA folding, rRNA processing, protein assembly, and subunit structure stabilization similar to what has been observed in other organisms (41)(42)(43)(44)(45)(46)(47)(48)(49). Intriguingly helicase activity was recently identified in ribosomes that could account for its ability to translate through downstream mRNA secondary structure (50).…”

“…The apparent strong association of a putative superoxide dismutase homolog in T. brucei mt ribosomes (i.e. the tagged protein pulled down the SSU) implies a possible antioxidant role, which may parallel the roles of the yeast Mrp1 and Rsm26 mt ribosomal proteins that also share similarities with proteins of the superoxide dismutase family (44).…”

“…For example, we found the T. brucei protein homolog of yeast and bovine MRPS29 that is the deathassociated protein 3 involved in apoptosis (8,44,52). Although the nature of apoptosis in this unicellular organism is uncertain, a role for mt ribosomes in this process cannot be disregarded because another T. brucei mt ribosome-associated protein (Tb927.7.2620) that we found belongs to the Bcl-2 family (53).…”

Properties of Human Mitochondrial Ribosomes

“…Recently, three different groups using proteomic approaches to analyze the proteins of bovine mitochondrial ribosomes (16 -27) have obtained sequence information for virtually all of the MRPs (about 80) ( Tables 1 and 2). Yeast MRPs are also available, for comparative purposes (28,29). The nomenclature system originally used to identify bovine MRPs was based on their electrophoretic properties (9).…”

Identification of phosphorylation sites in mammalian mitochondrial ribosomal protein DAP3

“…One of these, MRP-S29, more commonly known as DAP3, was first identified as a positive mediator of interferon-g induced cell death through a functional screen of HeLa cells transfected with an antisense cDNA library (Kissil et al 1995). Although it is historically linked to cell death, DAP3 is the only GTP-binding protein found in the small subunit of the mammalian and yeast mitochondrial ribosomes (Denslow et al 1991;Koc et al 2001a;Saveanu et al 2001). The presence of this protein accounts for the high specific GTP-binding affinity of small subunits of mitochondrial ribosomes (Denslow et al 1991).…”