SNARE‐mediated homotypic fusion in Golgi apparatus ribbon formation

Abstract

The ribbon‐like structure of the Golgi apparatus is thought to be the end result of homotypic fusion of cisternal membranes of ministacks, which are pre‐Golgi intermediate structures. Organization of ministack compartments for fusion is facilitated in part by the protein GRASP65 that links homotypic compartments of the ministacks and brings them into close contact. This process is called tethering. However, the Grasp65 protein alone is not sufficient to facilitate membrane fusion. Once the membranous compartments of the ministacks are in close proximity, a different set of proteins is believed to fuse the membranes into one continuous, ribbon‐like structure. The identity of these proteins is currently unknown. Soluble NSF attachment receptor (SNARE) proteins mediate membrane fusion in eukaryotic cells. A number of different SNARE proteins have been identified in Golgi membranes including Syntaxin 5, hBet1, Sec22, and Membrin. We believe that these four SNARE proteins along with GRASP 65 are sufficient to drive homotypic membrane fusion, possibly bringing about the fusion step that is required for Golgi ribbon formation. To test this hypothesis, I utilized a non‐Golgi membranous structure to model the activity of the proteins. Mitochondrial outer membranes provide an accessible model membrane to use in this case. I constructed plasmids that target the suspected Golgi SNAREs to the mitochondrial outer membrane. I then expressed these plasmids in HeLa cells along with GRASP 65 and assayed for membrane fusion using Fluorescence Recovery After Photobleaching (FRAP). As a result of my efforts, we are well on our way to determining the role of Golgi SNAREs in membrane fusion. This work was supported by the National Science Foundation's Research Opportunities for Undergraduates Program and the Mellon Institute of Carnegie Mellon University.