GRASP depletion–mediated Golgi destruction decreases cell adhesion and migration via the reduction of a5ß1 integrin

Ahat, Erpan; Xiang, Yi; Zhang, Xiaoyan; Bekier, Michael E.; Wang, Yanzhuang

To determine whether the reduction of α5β1-integrin level is due to decreased protein synthesis or increased degradation, we assessed integrin synthesis by measuring the incorporation of 35 S-labeled methionine and cysteine. Cells transfected with control or GRASP siRNAs were incubated in a medium containing 35 S-labeled methionine and cysteine for 1 h, and α5 and β1 integrins were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. The newly synthesized α5 and β1 integrins existed as single bands of 135 and 110 kDa, respectively ( Figure 6A ), corresponding to their immature ER forms. Notably, the amounts of α5 and β1 integrins were reduced by ∼50% following GRASP depletion ( Figure 6B) , consistent with the observation that the α5-and β1-integrin protein levels were reduced in GRASP-KO cells in a steady state ( Figure 5, A-E) .