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.
