A eubacterial origin for the human tRNA nucleotidyltransferase?

tRNA CCA-termini are generated and maintained by tRNA nucleotidyltransferases. Together with poly(A) polymerases and other enzymes they belong to the nucleotidyltransferase superfamily. However, sequence alignments within this family do not allow to distinguish between CCA-adding enzymes and poly(A) polymerases. Furthermore, due to the lack of sequence information about animal CCA-adding enzymes, identification of corresponding animal genes was not possible so far. Therefore, we looked for the human homolog using the baker's yeast tRNA nucleotidyltransferase as a query sequence in a BLAST search. This revealed that the human gene transcript CGI-47 (#AF151805) deposited in GenBank is likely to encode such an enzyme. To identify the nature of this protein, the cDNA of the transcript was cloned and the recombinant protein biochemically characterized, indicating that CGI-47 encodes a bona fide CCA-adding enzyme and not a poly(A) polymerase. This confirmed animal CCA-adding enzyme allowed us to identify putative homologs from other animals. Calculation of a neighbor-joining tree, using an alignment of several CCA-adding enzymes, revealed that the animal enzymes resemble more eubacterial ones than eukaryotic plant and fungal tRNA nucleotidyltransferases, suggesting that the animal nuclear cca genes might have been derived from the endosymbiotic progenitor of mitochondria and are therefore of eubacterial origin.

Purification and characterization of human cell--cell adhesion molecule 1 (C-CAM1) expressed in insect cells.

The cell--cell adhesion molecule 1 (C-CAM1) plays an important role as a tumor suppressor for prostate cancer. Decreased expression of C-CAM1 was detected in prostate, breast, and colon carcinoma. Reexpression of C-CAM1 in prostate and breast cancer cell lines was able to suppress tumorigenicity in vivo. These observations suggest that C-CAM1 may be used as a marker for cancer detection or diagnosis. To generate monoclonal antibodies specific to C-CAM1, we have overexpressed full-length human C-CAM1 in Sf9 cells using a baculovirus expression system. The protein was purified 104-fold using nickel affinity chromatography. About 0.4 mg purified C-CAM1 was obtained from 200 mg of infected cells. When the purified protein was digested with peptidyl-N-glycosidase, the apparent mobility of the protein on SDS--PAGE changed from 90 to 58 kDa, which is close to the molecular weight predicted from the cloned cDNA sequence. This observation suggests that C-CAM1 was glycosylated on asparagine residues when expressed in Sf9 cells. Western blotting and internal protein sequencing analysis confirmed that the purified protein is human C-CAM1. Biochemical and functional assays indicate that this protein expressed in Sf9 cells displays characteristics similar to those of native protein, including adhesion function and glycosylation modification. Using this protocol, sufficient quantity of this protein can be produced with purity suitable for monoclonal antibody generation and biochemical study.

Repair of tRNAs in metazoan mitochondria.

The integrity of 3'-ends of tRNAs is essential for aminoacylation and consequently for protein synthesis. The CCA-termini are generated and, if truncated by exonucleolytic activity, restored by tRNA nucleotidyltransferase. However, further truncations at the 3'-end can occur by exonuclease activity or during processing of overlapping tRNA primary transcripts in metazoan mitochondria. In the latter case, the upstream tRNA is released in a 3'-truncated form (lacking up to six bases) and subsequently completed. In human mitochondria, tRNA(Tyr)(missing the discriminator nucleotide A(73)) is completed by a discriminator adding activity followed by CCA addition. Since in vivo a high percentage of further 3'-terminally degraded human tRNA(Tyr)transcripts could be observed, it was tested in an in vitro system whether this repair mechanism for tRNA 3'-ends acts also on these further degraded tRNA versions. Additionally, 3'-truncated versions of two non-overlapping mitochondrial tRNAs (tRNA(Thr)and tRNA(Phe)) were examined. The results show that these transcripts can be repaired during incubation. A similar base incorporating activity was observed in mouse mitochondria, indicating that a repair mechanism for the 3'-end of several tRNAs exists in mitochondria of humans and possibly other metazoans which goes beyond the CCA addition.

Genetic and structural characterization of the human mitochondrial inner membrane translocase.

Translocation of nuclear-encoded mitochondrial preproteins is mediated by translocases in the outer and inner membranes. In the yeast Saccharomyces cerevisiae, translocation of preproteins into the matrix requires the membrane proteins Tim23, Tim17 and Tim44, which drive translocation in cooperation with mtHsp70 and its co-chaperone Mge1p. We have cloned and functionally analyzed the human homologues of Tim17, Tim23 and Tim44. In contrast to yeast, two TIM17 genes were found to be expressed in humans. TIM44, TIM23 and TIM17a genes were mapped to chromosomes 19p13.2-p13.3, 10q11. 21-q11.23 and 1q32. The TIM17b gene mapped to Xp11.23, near the fusion point where an autosomal region was proposed to have been added to the "ancient" part of the X chromosome about 80-130 MY ago. The primary sequences of the two proteins, hTim17a and hTim17b, are essentially identical, significant differences being restricted to their C termini. They are ubiquitously expressed in fetal and adult tissues, and both show expression levels comparable to that of hTim23. Biochemical characterization of the human Tim components revealed that hTim44 is localized in the matrix and, in contrast to yeast, only loosely associated with the inner membrane. hTim23 is organized into two distinct complexes in the inner membrane, one containing hTim17a and one containing hTim17b. Both TIM complexes display a native molecular mass of 110 kDa. We suggest that the structural organization of TIM23.17 preprotein translocases is conserved from low to high eukaryotes.