A Protein from a Parasitic Microorganism, Rickettsia prowazekii , Can Cleave the Signal Sequences of Proteins Targeting Mitochondria

Abstract

The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found to resemble the ␣ and ␤ subunits of mitochondrial processing peptidase (MPP), which cleaves the transport signal sequences of mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with ␤-MPP but barely contained any of the glycine-rich loop region characteristic of ␣-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover, RPP appeared to activate yeast ␤-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.The endosymbiont hypothesis for the origin of mitochondria in eukaryotes is now widely accepted, accompanied by a growing interest in evolution as entire genomic sequences are revealed from various organisms. According to this hypothesis, a free-living bacterium as an organelle progenitor once entered an anaerobic organism, which is thought to be an archaebacterial ancestor, and established a constitutive endosymbiotic relationship with the host cells, mainly to supply ATP (7,11,17). Modern mitochondria originated when the parasitic invader lost most of its own genome and began to depend on nuclearly encoded proteins for its biogenesis, although it retained control of the eukaryotic cell viability via metabolic and apoptotic pathways.Most mitochondrial proteins are encoded in the nucleus and synthesized by cytoplasmic ribosomes as preproteins with Nterminal presequences that are required for targeting to mitochondria (3, 12). These preproteins are unfolded and imported into the mitochondrial matrix across the double membrane through protein translocation machinery comprising a translocase on the outer mitochondrial membrane and a translocase on the inner mitochondrial membrane (20,24,26,27). Finally, the presequences are cleaved by a matrix-located metalloendopeptidase, i.e., mitochondrial processing peptidase (MPP) (10, 13, 16). Overall, this proteolytic processing is involved in maturation of the mitochondrial proteins and is essential for eukaryotic cell viability from unicellular (30) to multicellular (22) organisms. Therefore, the ␣ and ␤ subunits of MPP (␣-and ␤-MPP, respectively) tightly regulate the protease action and specifically cleave the preproteins.The genome sequences of the obligate intracellular parasitic bacteria rickettsiae (the agents that cause typhus) reveal gene profiles strikingly similar to those of mitochondria (2,18,23). Among ...

Glycine-Rich Loop of Mitochondrial Processing Peptidase α-Subunit Is Responsible for Substrate Recognition by a Mechanism Analogous to Mitochondrial Receptor Tom20

“…Interestingly, Kitada et al recently reported that the GRL is required only for the processing of long presequences: MPP enzyme containing α-MPP with the whole GRL deleted (positions 284-296) was still able to cleave yeast pMDH, albeit with a lower processing activity than the wild type. 23 Thus, the significant difference between the published results and our data is unexpected and indeed interesting.…”

Mitochondrion-Related Organelles in Eukaryotic Protists

“…With regard to the presequence processing enzyme in Giardia , it was shown that there is no α subunit, and that a single β-GPP is capable of cleavage of short leaders, similar to that described for a related proteinase from Rickettsia (61, 102). Interestingly, this monomeric giardial mitosomal processing peptidase is incapable of cleaving longer mitochondrial presequences (102).…”

Heterosubunit Composition and Crystal Structures of a Novel Bacterial M16B Metallopeptidase

“…Until recently, bacterial M16B enzymes have been suggested to be active in monomeric form based on the enzymology of Bacillus halodurans peptidase ppBH4 22 and Rickettsia prowazekii peptidase RPP. 23 On the other hand, the ppBH4-homologous BHP from another strain of B. halodurans has been recently reported to form an active homodimer. 18 The Gram-negative alginate-assimilating bacterium Sphingomonas sp.…”