Contacts between mammalian mitochondrial translational initiation factor 3 and ribosomal proteins in the small subunit

Abstract

Mammalian mitochondrial translational initiation factor 3 (IF3mt) binds to the small subunit of the ribosome displacing the large subunit during the initiation of protein biosynthesis. About half of the proteins in mitochondrial ribosomes have homologs in bacteria while the remainder are unique to the mitochondrion. To obtain information on the ribosomal proteins located near the IF3mt binding site, cross-linking studies were carried out followed by identification of the cross-linked proteins by mass spectrometry. IF3mt cross-links to mammalian mitochondrial homologs of the bacterial ribosomal proteins S5, S9, S10, and S18-2 and to unique mitochondrial ribosomal proteins MRPS29, MRPS32, MRPS36 and PTCD3 (Pet309) which has now been identified as a small subunit ribosomal protein. IF3mt has extensions on both the N- and C-termini compared to the bacterial factors. Cross-linking of a truncated derivative lacking these extensions gives the same hits as the full length IF3mt except that no cross-links were observed to MRPS36. IF3 consists of two domains separated by a flexible linker. Cross-linking of the isolated N- and C-domains was observed to a range of ribosomal proteins particularly with the C-domain carrying the linker which showed significant cross-linking to several ribosomal proteins not found in prokaryotes.

Identification and characterization of CHCHD1, AURKAIP1, and CRIF1 as new members of the mammalian mitochondrial ribosome

“…The siRNA knock-down of MRPS39 (PTCD3) decreased the activities of Complex III and Complex IV, possibly by directly affecting mRNA binding to the mitochondrial ribosome, as shown in our cross-linking experiments (Haque et al, 2011). In fact, PTCD3 was identified as one of the mRNA-binding proteins and IF3 mt interacting proteins located near the mRNA-binding path of the small subunit in crosslinking studies (Davies et al, 2009).…”

“…Our data not only confirms the association of PTCD3 with the small subunit, but also provides evidence that it is a bona fide ribosomal protein. This idea was recently reinforced by the observation that PTCD3 is one of the 28S subunit proteins that interacts with mitochondrial initiation factor 3 (Haque et al, 2011). The full-length bovine PTCD3 is 77.8 kDa with a pI of 6.0; it is possibly the largest protein, and one of the most acidic components of the 55S ribosome.…”

“…Previous work had indicated that PTCD3 was associated with the small subunit, while CRIF1 and ICT1 interacted with the large subunit (Koc and Spremulli, 2003; Richter et al, 2010; Haque et al, 2011; Kim et al, 2012). In the present analyses, CHCHD1 and AURKAIP1 signals overlapped with the MRPS29 signals, which were clearly detected in 28S and 55S fractions, indicating their association with the small subunit.…”

Mechanism of protein biosynthesis in mammalian mitochondria

“…The head of the 28S subunit lacks a number of the bacterial ribosomal protein homologs and appears to contain several proteins specific to mitochondrial ribosomes. To obtain insights into the protein neighbors of IF3 mt on the 28S subunit a chemical cross-linking procedure followed by mass spectrometry was undertaken [81]…”

Yeast PPR proteins, watchdogs of mitochondrial gene expression

“…36); PTCD3 has been identified as a small ribosomal subunit that cross-links to the initiation factor IF3. 36 It will be interesting to know whether interactions can be found between the yeast Dmr1 or Ppr3 and the corresponding yeast IF3 factors, and whether Dmr1 and Ppr3 co-sediment with the small ribosomal subunit in sucrose gradients of mitochondrial extracts.…”