Molecular mechanisms of cargo sorting in the early secretory pathway: role in diseases development.

Description of projects available to graduate students:
Research Projects available.
1. The regulation of cargo selection and export from the endoplasmic reticulum (ER). Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery, which includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. COPII associate with cargo and/or cargo receptors upon recruitment to the ER membrane by the activation of Sar1. Sar1 initiates cargo concentration and selection by concentrating cargo into morphologically identifiable tubular domains on the ER membrane. Subsequent addition of COPII components leads to vesicular export of the selected cargo. Utilizing a combination of biochemical and morphological assays which reconstitute the process of COPII mediated cargo selection and vesicle formation from the ER with purified protein components, the student will explore mechanisms which initiate control and regulate the process. The student will pursue preliminary data that already indicates a role for kinases in the regulation of ER export.

2. The regulation of cargo degradation in the ER.
A new experimental system is been developed to analyze the process of cargo protein degradation in the ER in a synchronized manner. Utilizing both in vivo and in vitro biochemical and morphological assays, the student will determine the identity of proteases involved in the process, the role of auto-phagocytosis in ER degradation, the regulation which is exerted on the degradation and the molecular mechanisms that divert cargo proteins from a productive folding pathway to degradation. The student will pursue preliminary data in which we have demonstrated diversion of a model cargo protein to ubiquitination and degradation in a synchronized manner both in vivo and in vitro.

3. Reconstitution of transport in the early secretory pathway in primary neurons. The endoplasmic reticulum (ER) is a polarized compartment. Newly synthesized proteins which translocate into the ER are inserted at the ribosome containing Rough ER and export from the compartment takes place from smooth ER membrane. In neurons, smooth ER membranes extend from the cell body into the distal processes. In preliminary studies we have begun to characterize the location of ER export sites. We are utilizing both in vivo and in vitro by reconstitution of export site formation in a neuronal cell-free assay using purified COPII components and primary mouse hippocampus derived neurons. ER export in these cells and the formation of Sar1 dependent tubular domains that can export cargo is found in both the cell body and dendrites. Using these techniques, the student will explore the possibility that ER export also occurs in the neuron axon in order to determine whether proteins are exported from the ER in a polarized manner. In addition, molecular mechanisms which mediate domain formation on the ER of neurons will be explored. Specifically, the role of cytoskeletal elements and molecular motors will be analyzed. In addition the selection and mobilization of signaling cargo molecules from the ER will be determined. Specifically, the role of transport in presenilin assisted signaling process will be analyzed. Presenilins are proteins which cycle between the ER and pre-golgi intermediates and are involved in the early onset of Alzheimer disease.

4. Cyctic finrosis transmembrane conductance regulator biogenesis:
Cystic fibrosis is an autosomal recessive human genetic disorder that is caused by the loss or disfunction of a plasma membrane cAMP regulated HCO3-/ CL- channel, the CF trans-membrane conductance regulator (CFTR). The most common mutated allele encodes for deletion of Phenylalanine in position 508 (D 508) in the CFTR protein, a mutation which leads to the production of an unstable protein. Mutated CFTR is not transported to the golgi complex and instead is degraded in the endoplasmic reticulum (ER). The sorting mechanisms that select mutated (and also 70% of wild type) CFTR for degradation or transport are largely unknown. Morphological and biochemical in vitro assays will be developed to define the sorting sites that operate between the ER and the golgi to select CFTR for transport or degradation. The in vitro assays will also be utilized to capture and isolate transport and degradation intermediates for molecular characterization and functional analysis.

Techniques graduate student will learn:
The student will learn: protein purification, DNA manipulation, recombinant protein production, Cell culture techniques, in vitro transport reconstitution assays (biochemical), immuno-fluorescent microscopy and in vitro morphological assays.