Molecular Dissection of the Secretory Pathway
My laboratory is investigating the intracellular trafficking, sorting and processing of polypeptide hormone precursors. These molecules require targeting to the correct organelle where they are "processed" to generate bioactive molecules. A major goal of our research is to determine how the structure of the Golgi apparatus is maintained so that different classes of cargo proteins are sorted to their appropriate cellular destinations. Understanding the details of this process is important because the Golgi apparatus undergoes fragmentation and re-assembly during normal cell division. However, in several autoimmune diseases the Golgi fragments irreversibly leading to cell death (apoptosis).
Two related approaches are being used to understand Golgi structure and function. The first involves determining the mechanism of secretory vesicle release from the Golgi apparatus. We have shown that vesicle budding from the trans-Golgi network (TGN) requires a small GTP-binding protein, ADP-ribosylation factor-1, (ARF1). In its GTP-bound state, ARF1 activates a lipid hydrolyzing enzyme phospholipase D (PLD) which is associated with Golgi membranes. PLD hydrolyzes phosphatidylcholine to generate phosphatidic acid (PA); the accumulation of PA appears to drive vesicle budding; in addition, PA is essential to maintain Golgi structure. We found that PA regulates production of the inositol phospholipid, phosphatidylinositol 4,5 bisphosphate (PIP2). In the absence of PA, the level of PIP2 in the Golgi apparatus drops rapidly resulting in fragmentation of the organelle. Golgi fragmentation occurs because several cytoskeletal proteins associated with the organelle including a specific form of spectrin bind to PIP2; in the absence of PIP2 synthesis, bIII-spectrin is phosphorylated resulting in its dissociation from the membrane. Currently, we are investigating the mechanism whereby various Golgi organelle proteins and lipid kinases interact to regulate Golgi structure.
Our second approach is to understand how the Golgi apparatus breaks down during apoptosis. We demonstrated that during cell death the proteases caspase-3 and -8 cleave several Golgi proteins implicated in vesicle trafficking and Golgi architecture. This leads to disruption of protein-protein interactions that tether vesicles to the Golgi apparatus and results in fragmentation of the organelle. Strikingly, a caspase cleavage fragment of one such molecule, p115, translocates into the cell nucleus and its expression alone is sufficient to induce apoptosis in otherwise healthy cells. Our results demonstrate a novel, uncharacterized interaction between the Golgi apparatus and nucleus and show that molecules previously thought to be restricted to protein trafficking also play a role in mediating the apoptotic response. Characterizing the mechanism of apoptotic Golgi fragmentation is a major goal of the laboratory.
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Selected References: 
R. Chiu, L. Novikov, S. Mukherjee, and D. Shields. A Caspase Cleavage Fragment of p115 Induces Fragmentation of the Golgi Apparatus and Apoptosis. J. Cell Biol. 159: 637- 648 (2002).
A. Siddhanta, A. Radulescu, M. C. Stankewich, J. S. Morrow and D. Shields. Fragmentation of the Golgi Apparatus: A Role for bIII Spectrin and Synthesis of phosphatidylinositol(4,5)bisphosphate. J. Biol. Chem. 278: 1957-1965 (2003).
Z. Freyberg, A. Siddhanta, and D.Shields “Slip, Sliding Away: Phospholipase D and the Golgi Apparatus” Trends Cell Biol. 13: 540-546 (2003).
D. Cai, M. Zhong, R. Wang, W. J. Netzer, D. Shields, H. Zhen3, S. S. Sisodia, D. A. Foster, F. S. Gorelick, H.Xu and P. Greengard., Phospholipase D1 corrects impaired bAPP trafficking and neurite outgrowth in FAD-linked PS1 mutant neurons. Proc. Natl . Acad. Sci USA. 103:1936-1940 (2006).
A. E. Radulescu, A. Siddhanta and D. Shields. A Role for Clathrin in Re-Assembly of the Golgi Apparatus. Mol Biol Cell. 18(1):94-105 (2007).
S. Mukerjee, R. Chiu, S. M. Leung and D. Shields. Fragmentation of the Golgi Apparatus: an early apoptotic event independent of the cytoskeleton. Traffic. 8(4):369-78 (2007).
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