Identification of GRASP65‐interacting proteins and characterization of their roles in Golgi cisternal stacking

Abstract

In vitro assays identified the Golgi peripheral protein GRASP65 as a Golgi stacking factor that links adjacent Golgi cisternae by forming mitotically regulated trans‐oligomers. Expression of non‐regulatable GRASP65 mutants in HeLa cells enhanced Golgi stacking in interphase and inhibited Golgi fragmentation during mitosis. Depletion of GRASP65 by small interference RNA (siRNA) reduced the number of cisternae in the Golgi stacks; this reduction was rescued by expressing exogenous GRASP65. Further experiments indicate that GRASP65 function is regulated by proteins in the cytosol. In this study, we have applied biochemical approaches to identify GRASP65‐interacting proteins. HeLa cell cytosol was subjected to fractionations using standard biochemical methods followed by affinity chromatography. Proteins that bound to GRASP65 were identified by mass spectrometry and further characterized. Our study has identified potential candidates that play essential roles in Golgi stack formation.