The use of BirA-BAP system to study the effect of US2 and US11 on MHC class I heavy chain in cells

- A method to study protein turn-over in cells is described.
- The assay is based on biotinylation of a tagged protein in cells expressing biotin ligase.
- Degradation of MHC proteins mediated by HCMV US2 and US11 could be observed by using this method.
- The assay can be used to study protein traffic and turn-over in cells.

Biotinylation has been extensively used for antibody tagging, affinity-based purification, and in protein/DNA-protein interaction studies. Here we describe the use of biotinylation to study the turn-over of proteins in cells. We use the prokaryotic biotin ligase (BirA) to biotinylate the human leukocyte antigen (HLA)-A2 (A2) heavy chain (HC), which was engineered to contain a biotin acceptor peptide (BAP). Controlled availability of biotin in combination with visualization using streptavidin-conjugated peroxidase made it possible to detect biotinylated BAP-A2. Further, we exploited the effects of human cytomegalovirus (HCMV) unique short (US) proteins US2 and US11 on the turn-over of BAP-A2 HC. The full-length BAP-A2 HC and its mutants lacking either the cytosolic tail (tail-less) or both the transmembrane and cytosolic regions (soluble) were expressed via recombinant adenoviruses (rAd). The effect of US2, US11 and a control HCMV protein US9, also expressed via rAd, on each of the BAP- A2 forms was assessed. Experiments using this system showed that US2 and US11 cause proteasome-mediated degradation of full-length BAP-A2 HC but only US2 could cause degradation of tail-less BAP-A2. The results demonstrate that the technique of biotinylation can be used to study protein turn-over in cells.