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

α5 Integrin is synthesized by the ER and transported through the Golgi to the plasma membrane. In the TGN, it is cleaved by PCs to become mature. To determine whether GRASP depletion reduces α5-integrin level by inducing ER-associated protein degradation (ERAD) or by affecting its maturation, we blocked newly synthesized α5 integrin in the ER by BFA treatment for 2 h and released it for indicated times in the presence of cycloheximide (CHX) and MG132 or monensin. MG132 inhibits ERAD, while monensin blocks the trafficking of α5 integrin from the Golgi stack to the TGN and thus inhibits α5-integrin cleavage. In this experiment, we determined the level of the full-length uncleaved form of α5 integrin on reducing gels. As GRASP KO cells have lower integrin levels, we exposed those gels longer, and thus all cell lines had a similar signal at the 0 time point to start with, and the reduction of the protein was assessed over time. As shown in Figure 5G , the decrease of full-length α5 integrin in GRASP-knockout cells was faster than in WT cells. The reduction of full-length α5 integrin could be the end result of one of two processes. One is ERAD; this possibility was ruled out, because the α5-integrin level was not rescued by MG132 ( Figure 5G , lane 5 vs. lane 4). The other possibility is that α5 integrin is cleaved by PCs in the TGN to mature; this explanation was supported by the inhibition of the reduction by monensin ( Figure 5G , lane 6 vs. lane 4). These results demonstrate that GRASP KO does not cause ERAD of α5 integrin; rather, it accelerates integrin trafficking and maturation, which is consistent with our previous observation in GRASPdepleted cells (Xiang et al., 2013) .