Our long range goal is to understand mechanistically the roles of membrane-bound and cytosolic proteins that direct the export of cargo from the endoplasmic reticulum (ER) of mammalian cells. A key step is the sorting of cargo into COPII vesicles. The events that underpin the physiological basis for cargo selection and concentration in COPII vesicles are unknown. To address this goal, we propose a complementary set of approaches to dissect the mechanism of cargo selection by focusing on the central role of the Sar1 GTPase.
Specific Aim I : Molecular and structural analyses of the mammalian Sar1 GTPase. We will examine the hypothesis that specific Sar1 effector domains direct the activity of Sar1 in coupling cargo selection to downstream target proteins involved in nucleotide exchange and hydrolysis, export site formation and concentration into COPII vesicles. These studies will involve site-directed mutagenesis and elucidation of the structure of Sar1 using x-ray crystallography and NMR.
Specific Aim II : Function of novel components of the Sar1-based cargo selection machinery. We will explore the hypothesis that currently unknown components of pre-budding complexes are part of a protein machine that coordinates cargo selection with other aspects of ER function to concentrate cargo into COPII vesicles. We will elucidate the composition of pre-budding complexes using mass spectrometry and characterize the function of novel components in ER export in vivo and in vitro.
Specific Aim III. Signals directing Sar1-dependent cargo selection. We will test the hypothesis that biosynthetic and endogenous proteins that recycle between the ER and post-ER compartments contain signals that serve as ligands to promote ER export. We will utilize well-developed in vitro cargo recruitment and budding assays to establish the minimal signal(s) necessary to promote efficient cargo export.
Specific Aim I V. Sar1 is a morphogenetic GTPase. We will examine the hypothesis that Sar1 is a 'morphogenic' GTPase involved in the generation of ER subdomains specialized in COPII vesicle formation. These studies will explore the novel possibility that membrane-associated receptors, microtubule motors and microtubules are critical components for normal packaging and export site selection in vivo. Each of the four specific aims addresses key issues circumscribing the function of the Sar1 GTPase in ER export. They will allow us to test current models to develop for the first time a mechanistic understanding of the crucial events in the cell biology of ER function in the normal and disease state.
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