News and Publications

Molecular and Structural Analysis of GTPase Regulation of Vesicular Transport in Health and Disease

W.E. Balch, C. Alory, Y. An, M. Asano, S.I. Bannykh, S. Béraud-Dufour, J. Matteson, B. Moyer, L. Page, H. Plutner, T. Sakisaka, K. Straley, J. Conkwright-Johnson, R. Wang

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 (1) vesicle coats and (2) 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.

During export from the first compartment of the secretory pathway, the endoplasmic reticulum, cargo recruitment to budding sites involves activation of the GTPase Sar1, a protein whose structure we recently solved at 1.7 Å by using x-ray crystallography (Fig. 1), by the protein mSec12, a guanine nucleotide exchange factor. This activation involves "exit codes" on cargo that help coordinate GTPase activation with vesicle formation through as yet unknown components. After activation, Sar1 recruits the coat complexes Sec23/24 and Sec13/31 from the cytosol to form a mature vesicle that separates from the endoplasmic reticulum and is targeted to the Golgi complex. During transit to the Golgi complex, the Sec23/24 coat complex functions as a GTPase-activating protein, returning the GTPase to the GDP-bound state, thereby triggering coat disassembly.

Further studies are aimed at solving the structure of the Sec12, Sec23/24, and Sec13/31 components of the export machinery. By identifying and characterizing the function of novel components involved in cargo sorting, we hope to gain critical insight into the basis for several inherited transport diseases, including cystic fibrosis, hereditary emphysema, and the neurodegenerative processes associated with amyloid disease.

Targeting and fusion to the Golgi complex require a different GTPase, Rab1. Rab proteins are critical 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 found that loss of function of the a-isoform of GDI is responsible for the inherited disease X-linked nonsyndromic mental retardation. The defect is a consequence of a mutation that makes GDI unable to bind prenyl groups present on Rab proteins. These results emphasize the importance of the GDI-Rab complex in neural events leading 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 the synapse. We solved the structure of the brain a-isoform of GDI at 1.04-Å resolution 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, recognition of the geranylgeranyl prenyl group found at the carboxyl termini of Rab proteins, and recognition of putative membrane receptors that involves a mobile effector loop found on the functional face of GDI in domain II.

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 single REP isoform (REP1) 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 role of REP and Rab in the 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 structural and molecular basis for 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
Allan, B.B., Moyer, B.D., Balch, W.E. Rab1 recruitment of p115 into a cis-SNARE complex: Programming budding COPII vesicles for fusion. Science 289:444, 2000.

Alory, C., Balch, W.E. Molecular basis for Rab prenylation. J. Cell Biol. 150:89, 2000.

Aridor, M., Balch, W.E. Kinase signaling initiates coat complex II (COPII) recruitment and export from the mammalian endoplasmic reticulum. J. Biol. Chem. 275:35673, 2000.

Aridor, M., Fish, K.N., Bannykh, S., Weissman, J., Roberts, T.H., LIppincott-Schwartz, J., Balch, W.E. The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly. J. Cell Biol. 152:213, 2001.

Bannykh, S.I., Bannykh, G.I., Fish, K.N., Moyer, B.D., Riordan, J.R., Balch, W.E. Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway. Traffic 1:852, 2000.

Béraud-Dufour, S., Balch, W.E. Structural and functional organization of ADP-ribosylation factor (ARF) proteins. Methods Enzymol. 329:245, 2001.

Chen, C.D., Huff, M.E., Matteson, J., Page, L., Phillips, R., Kelly, J.W., Balch, W.E. Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca²⁺ stabilization. EMBO J., in press.

Huang, M., Weissman, J.T., Plutner, H., Béraud-Dufour, S., Wang, C., Chen, W., Wilson, I.A., Balch, W.E. Crystal structure of Sar1-GDP at 1.7-Å resolution and the role of the N-terminus in ER export. J. Cell Biol., in press.

Moyer, B.D., Allan, B.B., Balch, W.E. Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis-Golgi tethering. Traffic 2:268, 2001.

Moyer, B.D., Balch, W.E. Structural basis for Rab function: An overview. Methods Enzymol. 329:3, 2001.

Moyer, B.D., Matteson, J., Balch, W.E. Expression of wild-type and mutant green fluorescent protein-Rab1 for fluorescence microscopy analysis. Methods Enzymol. 329:6, 2001.

Weissman, J.T., Aridor, M., Balch, W.E. Purification and properties of rat liver Sec23-Sec24 complex. Methods Enzymol. 329:431, 2001.

Weissman, J.T., Plutner, H., Balch, W.E. The mammalian guanine nucleotide exchange factor mSec12 is essential for activation of the Sar1 GTPase directing endoplasmic reticulum export. Traffic 2:465, 2001.

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