It would however be problematic to reconcile published functional data for both proteins if their N-terminal domains were oriented toward the lumen of the TGN. We aimed to distinguish between the various possible topologies through application of a Pristimerin trypsin protection analysis of CaBP7 and CaBP8 tagged with fluorescent protein variants either N- or C-terminally. We demonstrated that only C-terminal tags are afforded protection from cytosolic proteolysis. These data indicate that the Cterminus of both CaBP7 and CaBP8 must reside within the TGN lumen and that they do adopt a cytosolic N-terminal topology required for reported regulation of PI4K. A potential artefact of this assay relates to the fact that TA proteins are a special instance of type- II membrane proteins and it has been documented that appendage of protein tags at the C-terminus of TA-proteins can result in co-translational processing as typical type-II proteins. In the case of the TA protein cytochrome b5 it has been demonstrated that addition of up to 85 residues C-terminal to its TM helix still supports post-translational membrane insertion. Collectively these studies indicate that there may be additional determinants within TA protein sequences that are involved in selection for processing by post-translational mechanisms. To further characterise CaBP7 and CaBP8 membrane insertion we extended our topological analyses to examine accessibility of a ten residue c-myc epitope inserted within the C-terminal tail. This inclusion increased the C-terminal domain length of CaBP7 and CaBP8 to 20 amino acids and therefore they would still be expected to be processed only by dedicated TA protein membrane insertion mechanisms. Our data clearly indicate that the C-terminus of both reporter proteins reside within the lumen of sub-cellular organelles confirming the type-II topology suggested by our trypsin protection assays. That minimal perturbation to the length of the C-terminal tail of CaBP7 and CaBP8 permits a type-II membrane topology suggests post-translational processing by a tail-anchor specific mechanism. In this study we establish the existence of functional TM domains in CaBP7 and CaBP8 and describe the first example of TA protein targeting in the CaM related super-family of small EFhand Ca2+ -sensors. TA proteins are conserved throughout Pregnanolone evolution and may represent one of the most primitive membrane protein architectures. Proteins of this class may have evolved prior to functional translocon machineries which would be consistent with their ability to insert into membranes independently of this pathway. Interestingly, the CaBP protein sub-family seems to have appeared at a point in evolution that coincided with the development of vertebrate species an observation that renders the use of TA protein targeting in this family all the more unique.