Abstract
The first step of plant-specific protein O-glycosylation is the hydroxylation of proline residues catalyzed by prolyl 4-hydroxylases (P4Hs). Plant P4Hs are classified into two types; the type 1 and the type 2 P4Hs. Our previous study indicated that NtP4H2.2, which is a type 2 P4H in tobacco, is a peripheral membrane protein localizing in the lumenal side of the Golgi apparatus. The tox1 domain, which locates the C-terminal part of this protein, was demonstrated to have a membrane binding ability. Thus, it is possible that NtP4H2.2 associates with proteins localizing in Golgi membrane. We therefore searched proteins that associate with the NtP4H2.2.
Detergent treatment of microsomes prepared from tobacco BY-2 cells suggested that NtP4H2.2 is localized in the lipid raft. Triton X-100 insoluble fraction from the microsomes was treated with a cross-linker EGS. Immunoprecipitation of cross-linked NtP4H2.2 complex using a specific antibody allowed us to identify several candidates of NtP4H2.2 partners. We are currently characterizing them with mass-spectrometry and results of this analysis will be presented.
