Abstract
The first step of plant-specific protein O-glycosylation is the hydroxylation of proline residue catalyzed by prolyl 4-hydroxylases (P4Hs). To investigate this modification, we have been analyzing P4Hs in tobacco. This time we cloned three full-length cDNAs for type2 P4H (NtP4H2.1, NtP4H2.2, NtP4H2.3). All the encoded proteins contain N-terminal signal peptide, catalytic domain and C-terminal Tox1 domain, the function of which is unknown. Membrane fractionation analysis using a specific antibody for NtP4H2.2 revealed NtP4H2.2 distributes in fractions containing Golgi apparatus. NtP4H2.2 fused with GFP co-localized with a cis-Golgi marker protein in tobacco BY-2 cells. Almost all NtP4H2.2 was recovered in sedimentable fraction after the sonication of microsome. These results indicate type2 P4H is a membrane protein localized predominantly in the Golgi apparatus. We are currently investigating the function of the Tox1 domain and results of this analysis will be presented.