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 test the possibility that GRASP depletion may also affect the stability of α5β1 integrin, we blocked protein synthesis by CHX treatment and assessed the α5β1-integrin level over time. These proteins were stable, with no significant reduction within 36 h of CHX treatment, and were not affected by GRASP knockdown ( Figure 6C ) or knockout (Supplemental Figure 6 ). These results are consistent with previous reports that integrins are stable proteins in the cell (Lobert et al., 2010; Bottcher et al., 2012) . To further confirm these results by an alternative approach, we performed the same metabolic labeling and pulse-chase assay as described earlier. HeLa cells transfected with control or GRASP siRNAs were incubated in a medium containing 35 S-labeled methionine and cysteine for 1 h; chased for 12, 24, or 48 h; and lysed. Then α5 and β1 integrin were immunoprecipitated and analyzed by nonreducing SDS-PAGE and autoradiography. We found that, within 48 h, the degradation rates of both α5 and β1 integrin were not significantly affected by GRASP depletion (Figure 6 , D-F). Therefore, we conclude that GRASP depletion reduces the synthesis of α5β1 integrin but does not affect their stability.