News and Publications

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

W.E. Balch, C. Alory, Y. An, S.I. Bannykh, S. Béraud-Dufour, J. Conkwright-Johnson, J.-H. Kim, J. Matteson, L. Page, H. Plutner, T. Sakisaka, 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, that is, the endoplasmic reticulum, the Golgi complex, and the plasma membrane. 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 and targeting-fusion components to the nascent vesicle. The polymerization of these components 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, by the protein mSec12, a guanine nucleotide exchange factor. This event 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 system, returning the GTPase to GDP-bound state, thereby triggering coat disassembly.

Further studies are aimed at solving the structure of other 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 diseases.

Targeting and fusion of the coat complex II vesicle to the Golgi complex require a different GTPase, Rab1, that is recruited during vesicle budding. Rab1 is a member of a large family of related Rab GTPases (63 members) that act as molecular switches to regulate vesicle docking and fusion through the cyclical association of the Rab proteins with a large variety of different vesicular carriers. Each Rab GTPase acts at a different step in the exocytic and endocytic pathways, thereby defining the topologic organization of membranes in cells.

An important common effector that directs Rab recycling is the protein GDP-dissociation inhibitor (GDI), which forms a cytosolic complex with Rab proteins in their inactive, GDP-bound form. GDI binds Rab through the effector domain of the inhibitor (commonly referred to as switches I and II) and through C20 prenyl (geranylgeranyl) groups that are attached to the C termini of all Rab GTPases. The prenyl groups are essential for association of Rab with the membrane. During vesicle formation, the cytosolic GDI-Rab 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 components involved in association of a new vesicle with the actin-microtubule cytoskeleton and recruitment of tethering and fusion factors that direct the vesicle to the next compartment. After fusion, Rab is inactivated and is retrieved from membranes by GDI for subsequent reuse.

To address the mechanism of GDI function in vesicular traffic, we developed in vitro biochemical assays to study GDI-dependent Rab trafficking pathways and the molecular interactions that direct binding of Ras to GDI. In addition, using x-ray crystallography, we solved the structure of the brain a -isoform of GDI at 1.04-Å resolution. GDI is a 2-domain protein (Fig. 1). Using molecular genetic studies in yeast, we identified critical residues in domain I of GDI that are involved in Rab binding, determined the function of a "mobile effector loop" in domain II that directs GDI membrane receptors, and identified residues that facilitate binding of the geranylgeranyl prenyl groups to GDI.

Our recent elucidation of the structure of the GDI-geranylgeranyl lipid complex revealed that the lipid is bound in a shallow hydrophobic pocket beneath the domain I Rab-binding platform (Fig. 1). Intriguingly, the binding of lipid throws a molecular switch that alters the structure of the mobile effector loop in domain II, thereby releasing the GDI-Rab complex from the membrane. The structure of the GDI-lipid complex provides for the first time an explanation of inherited disease. We previously discovered that loss of function of GDI is responsible for X-linked nonsyndromic mental retardation. The defect in this disease is due to a mutation that prevents GDI from recognizing prenyl groups present on Rab proteins and thereby blocks recycling. The critical residue that is mutated in the disease, Leu92Pro, forms part of the lipid-binding pocket (Fig. 1). These results emphasize the importance of formation of the GDI-Rab complex through interaction with the lipid group in neural events leading the development of the brain and human intelligence.

Currently, we are focusing on the function of an evolutionarily and structurally related group of proteins involved in Rab prenylation (REP proteins). These proteins bind newly synthesized Rab and assist in the lipid modification of Rab proteins by the enzyme geranylgeranyl transferase II before delivering the newly prenylated Rab protein to the membrane. The loss of a single REP isoform (REP1) is the cause of choroideremia, a disease that leads to the degeneration of the retinal pigmented epithelium and loss of vision. We anticipate that structural, molecular, and physiologic analyses of the function of REP1 in the eye will provide important insights into the role of prenylation of specific Rab isoforms in maintaining the function of the retinal pigmented epithelium in vision.

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

Alory, C., Balch, W.E. Organization of the Rab-GDI/CHM superfamily: the functional basis for choroideremia disease. Traffic 2:532, 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. 20:6277, 2001.

Huang, M., Weissman, J.T., Béraud-Dufour, S., Luan, P., Wang, C., Chen, W., Aridor, M., Wilson, I.A., Balch, W.E. Crystal structure of Sar1-GDP at 1.7 Å resolution and the role of the NH₂ terminus in ER export. J. Cell Biol. 155:937, 2001.

Huang, M., Weissman, J.T., Wang, C., Balch, W.E., Wilson, I.A. Protein engineering for crystallization of the GTPase Sar1 that regulates ER vesicle budding. Acta Crystallogr. D Biol. Crystallogr. 58:700, 2002.

Nishimura, N., Plutner, H., Balch, W.E. The d -subunit of AP-3 is required for efficient transport of VSV-G from the trans-Golgi network to the cell surface. Proc. Natl. Acad. Sci. U. S. A. 99:6755, 2002.

Sakisaka, T., Meerlo, T., Matteson, J., Plutner, H., Balch, W.E. Rab-a ;GDI is regulated by a Hsp90 chaperone complex. EMBO J., in press.

Yoo, J.S., Moyer, B.D., Bannykh, S., Yoo, H.M., Riordan, J.R., Balch, W.E. Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway. J. Biol. Chem. 277:11401, 2002.