Investigating Novel Regulators Of Golgi Membrane Tubulation
The Golgi complex serves as a vital organelle from which proteins and membrane lipids are modified, sorted, and trafficked to various destinations. Mutations that cause defects in structural maintenance or membrane trafficking at the Golgi are commonly linked to neurodegeneration, metabolic disease, and reproductive disorders. Both structural maintenance and membrane trafficking rely on cooperative efforts of coated vesicles and membrane tubules. Although extensive information is available for membrane coated vesicle traffic, knowledge of membrane tubules remains comparably deficient. Understanding the regulatory mechanisms behind membrane tubules may help elucidate how Golgi tubule biogenesis can respond to varying physiological stimuli such as increased secretory loads. I utilized an siRNA library against all known and purported human kinases, or the kinome, in a high throughput, microscopy-based screen that identified proteins involved in Brefeldin A (BFA)-induced Golgi membrane tubulation. This screen successfully identified siRNAs that significantly inhibited or enhanced the effects of BFA-induced Golgi tubulation. Among the identified hits, I further characterized two inhibitory siRNA that targeted ProteinAssociating with the Carboxyl-terminal domain of Ezrin (PACE1) and diacylglycerol kinase [gamma] (DGK-[gamma]), and determined that they play important roles in maintaining intact Golgi ribbon structures through regulating Golgi membrane tubule biogenesis. I found that these proteins also facilitate Golgi reassembly and anterograde membrane trafficking of both soluble and transmembrane proteins, further buttressing the importance of membrane tubules in multiple, cellular processes. The results of my studies will be useful not only in contributing to the understanding of existing regulatory mechanisms behind membrane tubulation but also in discovering novel mechanisms. Further studies on membrane tubulation will help shed light on physiological functions and possibly lead to translational advances in diseases that depend on membrane tubulation and/or trafficking.
Golgi; tubules; pace1; dgkg; dagk; rnai
Brown, William J
Cerione, Richard A; Vogt, Volker M
Ph.D. of Biochemistry
Doctor of Philosophy
dissertation or thesis