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

In contrast to the reduced integrin synthesis, total protein synthesis was increased by GRASP depletion, while some other proteins at the cell surface, such as IGFR and TfR, were unaffected. The Note that the protein levels of α1, αV, and α6 did not change after GRASP depletion in contrast to the robust reduction of α5 and β1 integrins. (B) Schematic α5-integrin domain structure and epitopes of indicated antibodies. (C) GRASP knockout reduces the α5β1-integrin level. The full-length (FL), heavy-chain (HC), and light-chain (LC) forms of α5 integrin, β1 integrin, GRASP55, GRASP65, and actin were blotted. Note the reduced protein level of α5 integrin on all three blots as well as β1 integrin compared with that in WT cells. (D) Quantitation of C normalized with actin. Statistical analysis was performed by comparison with WT cells. (E) Western blot of α5 integrin on nonreducing (without DTT) and reducing gels (with DTT). Arrow and arrowhead indicate full length and light chain, respectively. (F) GRASP knockout reduces α5β1integrin level at the cell surface. GRASP-KO cells were surface biotinylated, this was followed by streptavidin pull-down and Western blotting of α5 integrin, β1 integrin, TfR, and actin. Note that only the highly glycosylated forms of integrins were pulled down; this was clearer with β1 integrin (top band). (G) GRASP knockout accelerates α5-integrin maturation. WT and GRASP-KO HeLa cells were pretreated with BFA for 2 h and washed out and then released into CHX for 0, 2, or 4 h or CHX for 4 h with MG132 or monensin. As GRASP-KO cells have a lower level of integrin level, we exposed those gels longer, so 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. (H) Western blot of indicated HeLa cells transfected with α5 integrin-GFP or GFP. α5-Integrin heavy chain (α5-integrin HC), β1 integrin, GFP, and actin were blotted. The reduced protein levels of α5 and β1 integrins in GRASP-KO cells were rescued by expressing α5 integrin-GFP, but not by GFP alone. (I) α5-Integrin expression rescues the decreased cell attachment of GRASP-KO cells. Cells transfected with indicated constructs were analyzed in an attachment assay, as described in Figure 1 . The quantitation results are shown. **, p < 0.01; ***, p < 0.001. FIGURE 6: GRASP depletion reduces α5-and β1-integrin synthesis but has no effect on their turnover. (A) GRASP depletion reduces protein synthesis of α5 and β1 integrin. An equal number of HeLa cells transfected with indicated siRNAs were labeled with Trans 35 S-Label [ 35 S] for 1 h. Immunoprecipitated α5β1 integrin was analyzed by gel electrophoresis and autoradiography. The protein synthesis rates of both α5 and β1 integrin decreased in GRASP-depleted cells. (B) Quantitation of A. Statistical analysis was performed by comparison with control (ctrl) siRNA-treated cells. *, p < 0.05; ***, p < 0.001. (C) Western blot of GRASP-depleted HeLa cells treated with CHX for the indicated times. At 72 h posttransfection with the indicated siRNA, cells were treated with 100 µM CHX for 0, 4, 8, 12, 24, and 36 h; lysed; and analyzed by Western blot for β1 integrin and p97 on the same gel. As GRASP knockdown cells have a lower level of integrins, we exposed those gels longer, so 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. (D) GRASP depletion does not increase α5-and β1-integrin deg