Open AccessKnockout of the Golgi stacking proteins GRASP55 and GRASP65 impairs Golgi structure and function
Author(s) -
Michael E. Bekier,
Leibin Wang,
Jie Li,
Haoran Huang,
Danming Tang,
Xiaoyan Zhang,
Yanzhuang Wang
Publication year - 2017
Publication title -
molecular biology of the cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.463
H-Index - 225
eISSN - 1939-4586
pISSN - 1059-1524
DOI - 10.1091/mbc.e17-02-0112
Subject(s) - golgi apparatus , biology , microbiology and biotechnology , transport protein , glycosylation , hek 293 cells , stacking , genetics , endoplasmic reticulum , cell culture , chemistry , organic chemistry
Golgi reassembly stacking protein of 65 kDa (GRASP65) and Golgi reassembly stacking protein of 55 kDa (GRASP55) were originally identified as Golgi stacking proteins; however, subsequent GRASP knockdown experiments yielded inconsistent results with respect to the Golgi structure, indicating a limitation of RNAi-based depletion. In this study, we have applied the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to knock out GRASP55 and GRASP65, individually or in combination, in HeLa and HEK293 cells. We show that double knockout of GRASP proteins disperses the Golgi stack into single cisternae and tubulovesicular structures, accelerates protein trafficking, and impairs accurate glycosylation of proteins and lipids. These results demonstrate a critical role for GRASPs in maintaining the stacked structure of the Golgi, which is required for accurate posttranslational modifications in the Golgi. Additionally, the GRASP knockout cell lines developed in this study will be useful tools for studying the role of GRASP proteins in other important cellular processes.