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Structural Biology of Vesicular Transport

Research Summary

Jonathan Goldberg’s research is focused on the underlying mechanisms of vesicular transport. His goal is to understand how protein sorting by transport vesicles propagates and maintains the organization of intracellular membrane systems.

Our research is focused on understanding the mechanisms that govern vesicular traffic in eukaryotic cells. Small diffusible vesicles mediate a tremendous flux of secretory and membrane cargo proteins among intracellular organelles, yet each organelle maintains its identity—a characteristic composition of resident protein catalysts and lipids. To understand how specificity is achieved in vesicle transport, we have focused on a molecular description of the processes of vesicle formation and cargo capture by vesicle coat proteins (COPs).

A molecular model of the COPII transport-vesicle cage...

Our lab applies biophysical methods, particularly x-ray crystallography, to determine the atomic structures of vesicle-associated proteins, and we relate structure to function through cell-free biochemical experiments that reconstitute aspects of the vesicle-budding process.

Vesicle Transport Out of the Endoplasmic Reticulum
Much has been learned in the past decade about the process of vesicle-mediated transport from the endoplasmic reticulum (ER) to the Golgi apparatus. The vesicles that form on the ER are coated with a polymeric complex termed COPII, which is composed of three cytosolic proteins: Sar1 G protein, Sec23/24, and Sec13/31. Together, these proteins are able to initiate vesicle budding, capture cargo molecules, and self-assemble into a 60-nm cage that sculpts the underlying membrane into a bud.

In early work, we addressed the mechanistic basis for the activation of Sar1 and related G proteins at specific membrane sites. The conversion of Sar1 from its GDP- to GTP-bound form involves a unique conformational switching mechanism that triggers the exposure of a membrane anchor—a hydrophobic N-terminal sequence—causing the G protein to translocate from the cytosol to the membrane. This switching mechanism is catalyzed by guanine nucleotide exchange factors (GEFs) that drive the conversion to the GTP form. A series of crystal structures establishes how a GEF binds to G protein and converts it to the GTP-bound state. Efficient catalysis by the GEF can occur only in proximity to the membrane, and this spatial restriction controls the sites at which vesicle budding is initiated.

The COPII coat proteins Sec23/24 and Sec13/31 are recruited sequentially to membrane-bound Sar1-GTP. Sec23/24, a flattened, bow tie–shaped molecule, interacts directly with the G protein and with an extensive area of the ER membrane. Only then can Sec13/31 be recruited, because its binding site involves a composite surface across the Sec23/24–Sar1 complex. This stepwise assembly process ensures an orderly budding reaction, such that Sec23/24 can begin its chief function of cargo selection before Sec13/31 arrives to initiate coat self-assembly into the spherical lattice that drives vesiculation. The visualization of this process emerged from a series of snapshots—crystal structures of discrete complexes along the assembly pathway—determined in our lab.

Structural and biochemical work, in collaboration with Randy Schekman (HHMI, University of California, Berkeley) showed how the COPII coat captures a diverse range of cargo membrane proteins during the budding reaction. The surface of the Sec24 subunit is pitted with binding sites (we have discovered half a dozen distinct sites to date) for a series of transport signals—short cytoplasmic sequences on the cargo proteins. Each binding site on Sec24  captures a different signal sequence and many cargo molecules share the same signal; therefore, a very large repertoire of cargo proteins is concentrated into COPII vesicles and departs the ER en route to the Golgi apparatus.

Architecture of Vesicle Cages
Early studies on clathrin established the concept of a vesicular coat as a mechanical device for budding vesicles. It is the driving energy of coat self-assembly, coupled to the membrane bilayer through direct protein–lipid interactions, which deforms the membrane into a bud. The complete molecular model of the COPII cage, built from 24 copies of the Sec13/31 crystal structure, is a 60-nm polyhedron with an open-lattice architecture that is distinct from the clathrin cage. This model serves as a framework for ongoing studies of COPII coat assembly and disassembly reactions, particularly involving the packaging of inordinately large molecules such as procollagen and chylomicron particles.

The three vesicular cages designed for eukaryotic life—clathrin, COPI, and COPII—are built from evolutionarily related proteins. Nevertheless, the clathrin and COPII cages have very distinct designs and symmetry, and their structures offer little clue about shared architectural principles. Strikingly, crystallographic analysis of the COPI cage protein reveals a structure that is intermediate in design between COPII and clathrin, helping to explain the distant architectural relationship among the three cages.

As of March 12, 2014

Scientist Profile

Investigator
Memorial Sloan-Kettering Cancer Center
Cell Biology, Structural Biology