GRASP55 is phosphorylated during mitosis. (A) GRASP55 schematic with phosphorylation sites indicated. Predicted phosphorylation sites (T222, T225, S245, T249, and T435) are indicated by asterisks. myr: myristoylated N-terminal glycine. (B) Detection of GRASP55 phosphorylation using a band-shift assay. Golgi membranes were either treated with buffer (lane 1) or mitotic cytosol (MC, lanes 2–4). Membranes were reisolated and further treated with calf intestine alkaline phosphatase (CIP) in the absence (lane 3) or presence (lane 4) of a general phosphatase inhibitor, β-glycerophosphate (β-GP). Note the increase in molecular weight of GRASP55 under mitotic conditions was reversed by CIP. (C) GRASP55 phosphorylation by ERK2. Golgi membranes were incubated with indicated proteins followed by Western blotting. Note that ERK2/MEK1 phosphorylated GRASP55 to a similar extent as MC. (D) The mitotic phosphorylation sites of GRASP55 are in the SPR domain. GST-tagged GRASP55 constructs were incubated with buffer (lane 1), or interphase (IC, lane 2) or mitotic (MC, lane 3) cytosol and analyzed by immunoblotting. Note that the SPR domain (213–454), but not the GRASP domain (1–212), exhibited a band shift after MC treatment, whereas deletion of aa 213–231 largely abolished the phosphorylation. (E) Mapping the phosphorylation sites of GRASP55. Potential sites (asterisks in A) in His-GRASP55 were mutated to alanines or glutamates and the purified proteins were analyzed as in D.