Development and characterization of a membrane-permeant GRASP65-mimetic peptide that inhibits Golgi unlinking and...

The Golgi complex is central to cellular homeostasis and serves as a key processing and sorting hub for protein trafficking. In many cell types, the Golgi complex is organized as interconnected stacks of cisternae, forming a structure known as the Golgi ribbon. This ribbon undergoes dynamic remodelling during physiological processes, such as cell division, and under pathological conditions, including cancer and neurodegeneration. A critical step in the unlinking of the Golgi ribbon involves the phosphorylation of the stacking protein GRASP65, which leads to the separation of the ribbon into individual stacks, a process necessary for the G2/M transition of the cell cycle. However, existing tools for selectively manipulating the GRASP65 role in ribbon organization are limited by non-specific effects or technical challenges. Here, we present the development and characterization of a membrane-permeable peptide, R₈-GRASP65-S277, derived from GRASP65 and containing the phosphorylation site Ser277, which is essential for Golgi unlinking. This peptide effectively inhibited Golgi unlinking and mitotic entry in several cell lines, including cancer models. In contrast, a control peptide with a non-phosphorylatable alanine substitution (R₈-GRASP65-S277A) showed no such effect, confirming the specificity of the tool. Furthermore, the R₈-GRASP65-S277 peptide reversed Golgi unlinking induced by the chemotherapeutic agent doxorubicin, demonstrating its utility in studying stress-induced Golgi disassembly. These findings establish the R8-GRASP65-S277 peptide as a specific, potent, and scalable tool for probing the molecular mechanisms of Golgi unlinking, its regulation of cell cycle progression, and its potential contributions to pathological states.