News and Publications

Chemical Biology of Protein Traffic

The broad objective of our research program is to define the molecular basis for protein traffic through the secretory pathway of eukaryotic cells. We use chemical, structural, and biological approaches.

The eukaryotic cell is highly compartmentalized; each compartment of the exocytic and endocytic pathways defines a unique chemical landscape in which protein function is modulated. Movement between these compartments 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. A large number of conformational diseases, including amyloid diseases, are a consequence of dysfunctions in protein traffic.

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 a ligand to initiate a signal transduction pathway that leads to the assembly of vesicle coat complexes. Vesicle-mediated transport is regulated by a diverse group of small GTPases belonging to the Ras superfamily. Each of these molecules acts as a "molecular sensor" to regulate different steps in the reversible assembly of vesicle coats and targeting-fusion complexes to ensure the vectorial transport of cargo between distinct intracellular compartments. Activation of these GTPases to the GTP-bound form most likely kinetically regulates the sequential and specific recruitment of coat and targeting-fusion components to nascent vesicles. 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 solved at 1.7 Å by using x-ray crystallography, in collaboration with I.A. Wilson, Department of Molecular Biology. Activation occurs via 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. In collaboration with J. Yates, Department of Cell Biology, we are using multidimensional mass spectroscopy to identify and characterize the function of novel components involved in cargo sorting and thus elucidate the proteomics of cargo transport. After GTPase activation and cargo selection, Sar1 recruits the coat complexes Sec23/24 and Sec13/31 from the cytosol to form a mature vesicle that separates from the endoplasmic reticulum. Through this approach we hope to gain critical insight into the basis for several inherited transport diseases, including cystic fibrosis and hereditary emphysema, that are a consequence of defective export from the endoplasmic reticulum. Knowledge of these pathways will enable the development of small-molecule chemical chaperones to ameliorate disease.

After vesicles are formed, targeting and fusion of transport vesicles to downstream compartments require a different GTPase, Rab1. Rab1 is a member of a large family of related Rab GTPases (63 members) that act as molecular switches that assemble complexes involved in vesicle tethering and fusion (Fig. 1). Use of a bioinformatics approach revealed that each Rab GTPase executes targeting and fusion at a different step in the exocytic and endocytic pathways, thereby defining the topological organization of membranes in differentiated cells.

An important common effector that directs Rab recycling is the protein GDP-dissociation inhibitor (GDI), which forms a cytosolic complex with Rab proteins. GDI binds Rab through the effector domain of the inhibitor (commonly referred to as switches I and II) and through C20 prenyl (geranylgeranyl) groups found at the C termini of all Rab GTPases. The prenyl groups are essential for association of Rab with the membrane. Using x-ray crystallography, we solved the structure of the brain α-isoform of GDI at 1.04-Å resolution. Using molecular genetic studies in yeast, we identified critical residues that are involved in Rab binding, residues that function as a mobile effector loop that directs GDI to membrane receptors containing the Hsp90 chaperone complex, and residues that facilitate binding of the geranylgeranyl prenyl groups to GDI.

The structure of the GDI-geranylgeranyl lipid complex revealed that the lipid is bound in a shallow hydrophobic pocket beneath the Rab-binding platform. The binding of lipid throws a molecular switch that alters the structure of the mobile effector loop, thereby releasing the GDI-Rab complex from the membrane. This change in conformation is required for Rab-mediated steps in neurotransmission, because mutation of Leu92, a key residue in the lipid-binding pocket, is responsible for X-linked nonsyndromic mental retardation.

In addition to GDI, we are focusing on the function of REP proteins, an evolutionarily and structurally related group of proteins involved in Rab prenylation. REP proteins bind newly synthesized Rab through the structurally conserved Rab-binding domain and assist in the prenylation of Rabs. 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. Structural, molecular, and chemical analyses of REP1 function in differentiated retinal pigmented epithelium cultured in vitro are providing important insight into the role of specific Rab isoforms in phagocytosis of rod outer segments by cells in the epithelium, an important function for maintaining normal vision.

Although protein traffic involves vesicle formation and targeting, proteins implicated in a wide range of diseases have mutations that disrupt the conformation (folding) and, as a consequence, the trafficking patterns of the proteins within the chemical landscape of exocytic and endocytic compartments and the extracellular milieu. Analysis of the mechanisms of dysfunction of some of these proteins, including plasma gelsolin (familial amyloidosis Finnish type), ß-glucosidase (Gaucher disease), and transthyretin (familial amyloid polyneuropathy I), done in collaboration with J. Kelly, Department of Chemistry, is providing important insight into these diseases. Analysis of the chemical and biological basis for these defects will provide an understanding of the general principles of reversible regulation of organelle structure and function during cell growth and differentiation.

Publications

Alory, C., Balch, W.E. Molecular evolution of the REP/GDI superfamily. Mol. Biol. Cell, in press.

Alory, C., Balch, W.E. Molecular and structural organization of Rab GTPase trafficking networks. In: Handbook of Cell Signaling. Bradshaw, R.A., Dennis, E.A. (Eds.). Academic Press, San Diego, in press.

An, Y., Shao, Y., Alory, C., Matteson, J., Sakisaka, T., Chen, W., Gibbs, R.A., Wilson, I.A., Balch, W.E. GDI-Rab GTPase recycling. Structure (Camb.) 11:347, 2003.

Beraud-Dufour, S., Balch, W. A journey through the exocytic pathway. J. Cell Sci. 115:1779, 2002.

Kelly, J.W., Balch, W.E. Amyloid as a natural product. J. Cell Biol. 161:461, 2003.

Sakisaka, T., Meerlo, T., Matteson, J., Plutner, H., Balch, W.E. Rab-αGDI activity is regulated by a Hsp90 chaperone complex. EMBO J. 21:6125, 2002.

Sawkar, A.R., Cheng, W.C., Beutler, E., Wong, C.H., Balch, W.E., Kelly, J.W. Chemical chaperones increase the cellular activity of N370S ß-glucosidase: a therapeutic strategy for Gaucher disease. Proc. Natl. Acad. Sci. U. S. A. 99:15428, 2002.