News and Publications

Molecular and Structural Analysis of GTPase Regulation of Vesicular Transport

H. Plutner, J. Matteson, M. Aridor, S.I. Bannykh, N. Nishimura, K. Zeng, P. Luan, C. Alory, B. Allan, J. Weissman, B. Moyer, C.D. Chen, W.E. Balch

The broad objective of our research program is to define the molecular basis for GTPase function in membrane transport through the secretory pathway of eukaryotic cells. We use morphologic, biochemical, and structural (x-ray crystallography) approaches. Movement involves the activity of both anterograde and retrograde transport vesicles. These vesicles maintain a balance of membrane flow between organelles and recycle integral membrane components of the transport machinery. Vesicle-mediated transport is regulated by a diverse group of small GTPases belonging to the Ras superfamily. Each of these molecules serves as a GTP-based "molecular sensor" to regulate different steps in the reversible assembly of vesicle coats and of targeting and fusion complexes to ensure the vectorial transport of cargo between distinct intracellular compartments.

How do GTPases mediate formation of vesicle carriers? Transport through the secretory pathway involves a selective mechanism in which cargo molecules are concentrated into vesicles and resident proteins are excluded. Current evidence suggests that cargo may function as ligands to initiate a signal transduction pathway that leads to the assembly of vesicle coat complexes. Linked to each cargo-sorting event is the activation of a GTPase to the GTP-bound form, thereby kinetically regulating the sequential and specific recruitment of coat components to membranes. The polymerization of these activated coat complexes into a molecular lattice drives vesicle budding (Fig. 1).

After budding, the coat complex rapidly disassembles in response to hydrolysis of GTP, returning the GTPase to the GDP-bound state and preparing the vesicle for membrane fusion. During export from the first compartment of the secretory pathway, the endoplasmic reticulum, cargo recruitment to budding sites involves activation of the GTPase Sar1. This event requires "exit codes" on cargo that direct GTPase activation through currently unknown components. By determining and characterizing the function of novel components involved in cargo sorting, we hope to gain insight into a variety of inherited transport diseases, including cystic fibrosis, familial hypercholesteremia, and the neurodegenerative processes associated with Alzheimer's disease.

Whereas Sar1 regulates coat assembly on the endoplasmic reticulum, Rab proteins are crucial GTPase molecular switches that regulate vesicle fusion through cyclical association with vesicular carriers. This cycle is directed by the protein GDP-dissociation inhibitor (GDI), which forms a soluble complex with Rab proteins in their inactive, GDP-bound form. The GDI-Rab cycle is central to both exocytic and endocytic pathways. The cytosolic complex first delivers Rab to newly forming vesicles, where Rab is activated to the GTP-bound form. Activation is thought to coordinate the assembly of a protein complex involved in vesicle fusion to downstream compartments. After fusion, Rab is inactivated and is retrieved from membranes by GDI for subsequent reuse. Attesting to the central role of GDI in membrane transport, we recently found that loss of function of the -isoform of GDI is responsible for the inherited disease X-linked nonsyndromic mental retardation. These results emphasize the importance of the GDI-Rab complex in neural events that lead to the development of the brain and human intelligence.

To address the mechanism of Rab and GDI function, we developed in vitro assays to study biochemically Rab-dependent events that mediate transport from the endoplasmic reticulum to the Golgi complex and release of neurotransmitters at synapses, and we determined the structure of the brain -isoform of GDI by using x-ray crystallography. Using molecular genetic studies in yeast, we identified critical residues in subdomains of GDI that are involved in Rab binding and recognition of putative membrane receptors.

To expand our understanding of structure-function relationships, we are focusing on the structures of the native GDI-Rab complex and an evolutionarily related group of proteins involved in Rab prenylation (REP proteins). These proteins bind newly synthesized Rab and posttranslationally modify Rab proteins at the carboxyl-termini with prenyl lipids before delivering the modified proteins to the membrane. Prenylation is crucial for Rab function. Indeed, the loss of a REP isoform is the cause of the defect in choroideremia, a disease that leads to the degeneration of the retinal pigmented epithelium and loss of vision. Combined structural and molecular analysis of REP should provide important insight into the roles of REP and Rab in maintenance of epithelial cell polarity in the eye.

The biochemical mechanisms fundamental to vesicle fission and fusion are evolutionarily conserved across a wide spectrum of biological processes, including constitutive secretion, neurotransmission, and the regulated release of hormones from endocrine and exocrine cells. Understanding the signaling cascades that lead to GTPase activation and inactivation will provide insight into the general principles that regulate the structure and function of secretory organelles during cell growth and differentiation.

PUBLICATIONS

Aridor, M., Weissman, J., Bannykh, S.I., Nuoffer, C., Balch, W.E. Cargo selection by the COPII budding machinery during export from the endoplasmic reticulum. J. Cell Biol. 141:61, 1998.

Bannykh, S.I., Balch, W.E. Selective transport of cargo between the endoplasmic reticulum and Golgi compartments. Histochem. Cell Biol. 109:463, 1998.

Bannykh, S.I., Nishimura, N., Balch, W.E. Getting into the Golgi. Trends Cell Biol. 8:21, 1998.

D'Adamo, P., Menegon, A., Nigro, C.L., Grasso, M., Gulisano, M., Tamanini, F., Bienvenu, T., Gedeon, A.K., Oostra, B., Wu, S.-K., Tandon, A., Valtorta, F., Balch, W.E., Chelly, J., Toniolo, D. GDI1 is responsible for X-linked mental retardation. Nature Genet. 19:134, 1998.

Nuoffer, C., Wu, S.-K., Dascher, C., Balch, W.E. MSS4 does not function as an exchange factor for Rab in endoplasmic reticulum to Golgi transport. Mol. Biol. Cell 8:1305, 1997.

Rowe, T., Balch, W.E. Bridging the gap by AAA ATPases. Nature 388:20, 1997.

Rowe, T., Dascher, C., Bannykh, S.I, Plutner, H., Balch, W.E. Role of vesicle-associated syntaxin 5 in the assembly of pre-Golgi intermediates. Science 279:696, 1998.

Tandon, A., Bannykh, S.I., Kowalchyk, J.A., Banerjee, A., Martin, T.F.J., Balch, W.E. CAPS (mammalian UNC-31) regulates norepinephrine but not glutamate release from semi-intact synaptosomes. Neuron 21:147, 1998.

Tandon, A., Tan, P.K., Bannykh, S.I., Banerjee, A., Balch, W.E. Neurotransmitter release from semi-intact synaptosomes. In: Methods: A Companion to Methods in Enzymology. Academic Press, Miami, FL, in press.

Wu, S.-K., Luan, P., Matteson, J., Zeng, K., Nishimura, N., Balch, W.E. Molecular role for the Rab binding pocket of guanine nucleotide dissociation inhibitor (GDI) in endoplasmic reticulum to Golgi transport. J. Biol. Chem., in press.