BMC Genomics 2005, 6:125 http://www.biomedcentral.com/1471-2164/6/125 Table 1: Predicted mitochondrial signal for rTSβ and γ proteins Program Mitoprot TargetP PSORTII MITOPRED rTSγ 0.9382 0.719 26.1% 92.3% rTSβ 0.0117 0.077 8.7% 0 Reference [22] [23] [24] [25,26] (including mitochondria and lysosomes) were prepared from CCRF-CEM human cells, and their proteins were resolved by electrophoresis and analyzed for rTS protein expression (Fig. 3A). The results indicate that two rTS pro- teins with apparent molecular mass of 52.7 ± 1.8 and 47.6 ± 0.7 kDa (approximately corresponding to the difference in the predicted molecular mass of the rTSβ and γ iso- forms, respectively) are present. There was a preferential distribution of the higher molecular mass species in the organellar fraction as compared to the cytosolic fraction. The presence in the organellar fraction of both lysosomal (LAMP-1) and mitochondrial (MnSOD) marker proteins, however, did not allow us to conclude that rTS proteins are present in the mitochondria, rather than in the lyso- somes. To resolve this, we partially separated lysosomes from mitochondria using a 5–20% iodixanol gradient [8,9](Fig. 3B). The protein profile of the iodixanol gradi- ent shows a peak centered on fractions 14–15 with a pro- nounced shoulder in fractions 10–12. GDH activity appears in fractions 12–18 with a peak at fraction 15, indi- cating the presence of mitochondria in these fractions. The shoulder in the protein profile (fractions 10–12) that lacks GDH activity suggests the presence of non-mito- chondrial organelles, including lysosomes. This sugges- tion was confirmed by Western blotting of the gradient fractions for MnSOD and LAMP-1 markers (Fig. 3C). Analysis of the distribution of rTSβ in the gradient indi- cates that both the rTSβ and γ species co-localize with MnSOD, but not LAMP-1, conclusively demonstrating their presence in mitochondria, but not lysosomes. Discussion Functional implications for the rTS gene based on its mitochondrial location It has recently been determined that rTSβ is an enzyme responsible for the synthesis of small signaling molecules involved with the down-regulation of thymidylate syn- thase as cells enter growth arrest in vitro [6]. The signaling associated with rTSβ was also shown to act intercellularly [6]. Sequence analysis of the rTS gene suggested the pres- ence of a mitochondrial leader sequence that would be expected to increase the length of rTSβ from 416 to 443 amino acids in rTSγ with an expected change in molecular mass from 46,892 to 49,742 Da. Based upon the predic- tion of a mitochondrial leader sequence, we evaluated whether there is a mitochondrial form of the rTS protein. The results shown in Fig. 3 indicate that this is the case, with two species being present in the mitochondria. These two protein species have apparent molecular masses of 52.7 ± 1.8 and 47.6 ± 0.7 kDa. The smaller species corre- sponds well to the predicted size expected for rTSβ with the mitochondrial leader sequence cleaved off, while the larger species differs by 3 kDa from the predicted molecu- lar mass for the isoform with the leader sequence. The amounts of rTSγ and rTSβ in the combined cytoplasmic and mitochondrial fractions differ from that expected to result from combining equal amounts of each fraction. The increased abundance of rTSβ, relative to rTSγ in the combined fraction may be the result of in situ cleavage of the rTSγ mitochondrial leader sequence by cytosolic pro- teases, which has been observed to occur in yeast cytosol [10], although transfer of protein may also contribute to the change in signal strength as the MnSOD signal is weaker in the lane with the combined fractions than in the lane loaded with just the organelle protein. Although the migration of proteins in SDS-PAGE gives only approxi- mate estimation of molecular masses, there is a possibility that other post-translational modifications may contrib- ute to this discrepancy. The co-localization of rTSβ and rTSγ with GDH and MnSOD after subcellular fractiona- tion strongly suggests that the extended N-terminal sequence serves as the leader sequence for mitochondrial location of the protein and that it is likely this is partially cleaved to generate the β isoform, once rTSγ is transported into the mitochondria. The presence of the rTSβ protein in mitochondria raises the question of what role this enzyme and the rTS signaling pathway may play there. Mitochon- dria are a major site for folate metabolism in mammalian cells [11]. Thymidylate synthase is also found within mitochondria [12], despite the absence of a canonical mitochondrial leader sequence, and the relationship of rTS signaling to thymidylate synthase and folate metabo- lism may ultimately provide the explanation for this phe- nomenon. Recent evidence indicates that treatment of cells with an rTSβ signaling mimic can affect the cytoskel- eton and cause down-regulation of TYMS [10,13]. The co- localization of rTS and thymidylate synthase in the mito- chondria may indicate that rTSβ, in addition to its role in intercellular signaling, also provides intracellular signals that regulate thymidylate synthase levels in the mitochondria. Since the extended N-termini of rTS are conserved from bacteria to human, we believe that the rTSγ form of the rTS gene represents the ancestral form of this gene, while rTSβ, which seems to be the predominantly expressed form in the cytosol, represents an isoform that appeared later dur- ing evolution and came to co-exist with the ancestral iso- form, at least in Homo sapiens. Based on a recent study Page 5 of 10 (page number not for citation purposes)