Biophysical, Structural, and Cellular Dissection of COPI-Dependent Retrograde Trafficking Using a Coronavirus Toolkit

PROJECT SUMMARY The secretory pathway is responsible for the biogenesis of soluble and membrane proteins involved in communication, energy transduction, nutrient uptake, and defense. These proteins are synthesized in the endoplasmic reticulum (ER) and then trafficked to Golgi and other organelles such as the plasma membrane. This trafficking causes ER stress by accidental exodus of ER-resident proteins such as UDP-glucuronyl transferases (UGT’s). These are type I membrane proteins (T1MP’s) responsible for modifications of lipid hormones and of analgesics acetaminophen and morphine. These ER-resident T1MP’s display a dibasic sequence (Lys-x-Lys-x-x or Lys-Lys-x-x; x=any amino acid) in their cytosolic tail for ER-retrieval by the coatomer protein I complex (COPI). The α and β’ subunits of this hetero-heptameric complex bind this dibasic sequence to initiate T1MP packaging into vesicles originating from post-ER compartments such as cis-Golgi. These COPI coated vesicles traffic and deliver the T1MP proteins back to ER to restore secretory balance. However, the atomic principles underlying T1MP binding, release, and selective interactions with α and β’COPI subunits are not well understood. This is a critical knowledge-gap as T1MP release and escape from COPI modulate T1MP trafficking, post-translational modifications, and T1MP functions. COPI dysfunction has been implicated in a variety of disorders related to development, auto-immunity, and cancers. Our long-term objective is to gain fundamental insights into COPI-dependent retrograde trafficking of T1MP’s and the underlying atomic-level factors responsible for COPI dysfunction in diseases. In this grant, we will elucidate mechanistic insights into COPI recruitment, release, and T1MP post-translational modifications utilizing the coronavirus (CoV) spike protein, a T1MP with a dibasic Lys-x-His-x-x sequence, as a new model system. This dibasic sequence ensures COPI-dependent retrograde delivery of the spike from Golgi to the viral progeny assembly site in ER-Golgi intermediate compartment (ERGIC). In Aim 1, we will elucidate the atomic details of conformational modulation of COPI-spike interactions. In Aim 2, we will determine the principles that govern release from COPI and subsequent post-translational modifications of the spike. In Aim 3, we will elucidate the atomic basis of COPI subunit selectivity for the spike protein. These investigations will expand on a toolkit of spike mutants with modified COPI interactions, as recently published by our group. We will integrate structural approaches in X-ray crystallography, NMR, and Rosetta modeling with biophysical tools and cellular assays of secretory trafficking to gain unprecedented insights into fine modulation and conformational regulation of COPI-spike interactions. The innovative use of the spike protein as a T1MP model system will yield novel insights into fundamental secretory trafficking. These data will simultaneously opening avenues for the development of targeted therapeutics for COPI-selective disorders and for a deeper understanding of CoV assembly and processing of CoV vaccines.