Mammalian Golgi structure in ceramide trafficking, sphingomyelin synthesis, and cellular stress

The eukaryotic Golgi complex plays a central role in processing and sorting of cargo in the secretory pathway. Here, proteins destined for secretion or delivery to the plasma membrane are post-translationally modified and sorted into vesicles, which are then transported to target membranes. In addition to these functions, the Golgi also plays an important role in sphingolipid biosynthesis. The mammalian Golgi apparatus is composed of multiple stacks of cisternal membranes that are organized laterally into a ribbon-like structure and is held at a juxtanuclear location by microtubule association. The stacks are polarized, and cargo moves through the organelle in a cis-to-trans direction. In addition, high voltage electron microscopic images reveal that trans-Golgi membranes come in close apposition with specialized endoplasmic reticulum (ER) membranes to form ER-trans-Golgi contact sites, which are thought to facilitate lipid transfer from the ER directly to the trans-Golgi. The Golgi ribbon structure is unique to vertebrate cells as lower eukaryotic cells lack this elaborate architecture. This complex structural architecture of the mammalian Golgi ribbon suggests potential additional functions. In this thesis, I propose that Golgi structure, including the ribbon and the ER-trans-Golgi connections, is important for ceramide trafficking and sphingomyelin (SM) synthesis.;A protein called CERT (ceramide transfer protein) delivers ceramide from its site of synthesis in the ER to the enzyme SM synthase at the trans-Golgi for SM synthesis. CERT interacts with both ER and Golgi membranes, and may function at the ER-trans-Golgi contact sites. Some Golgi structural perturbations, including ministacks formed by nocodazole treatment, reduced de novo SM synthesis as well as CERT's colocalization with Golgi markers. However, other Golgi morphologies, including vesicles generated by ilimaquinone treatment were compatible with efficient ceramide trafficking and SM synthesis. This suggests that certain Golgi morphologies together with CERT may promote specific ER-Golgi interactions for efficient delivery of ceramide for SM synthesis. Under cellular stress, caspase activation and increased ceramide levels can lead to Golgi ribbon disassembly and loss of ER-trans-Golgi contact sites. Prolonged stress that cannot be repaired usually results in apoptosis. Interestingly, Golgi disassembly during proapoptotic signaling by tumor necrosis factor (TNFalpha) and anisomycin was associated with decreased levels of CERT at the Golgi region. This was accompanied by a reduction of full length CERT protein and SM synthesis, in a caspase-dependent manner. CERT was cleaved by caspases-2, 3, and 9 in vitro. We characterized and mapped cleavage of CERT by caspase-2, since caspase-2 is partially localized at the Golgi and activation of the Golgi pool of caspase-2 may play an important role in integrating stress signaling pathways at the Golgi. Truncated versions of CERT corresponding to fragments generated by caspase-2 cleavage were mislocalized and non-functional. While full length CERT protein rescued SM synthesis in cells depleted of endogenous CERT by RNA interference, the N-terminal fragment of CERT did not. Thus, it is likely that during cellular stress, disassembly of Golgi structure together with inactivation of CERT by caspases causes a reduction in ceramide trafficking and SM synthesis. However, an important question remains unanswered: whether ER-trans-Golgi contact sites are upstream targets of stress signals that lead to increased ceramide levels and caspase activation, or if altered ceramide trafficking is downstream of Golgi disassembly. Regardless, it is clear that Golgi ribbon structure, including ER-trans-Golgi contact sites, is exquisitely sensitive to perturbation, making this organelle an ideal platform to sense cellular stress and integrate signals that determine cell survival or cell death.