Fig. 1: The conserved C-terminus determines S protein trafficking and coatomer interactions.
From: A single C-terminal residue controls SARS-CoV-2 spike trafficking and incorporation into VLPs
A Upper panel: Schematic of bidirectional trafficking of S in the secretory pathway. The newly synthesized S protein (yellow) in the ER (endoplasmic reticulum) is packaged in vesicles and trafficked to the ERGIC (endoplasmic reticulum-Golgi intermediate complex; steps 1, 2), and delivered to the cis-Golgi (steps 3, 4). COPI-coated vesicles (pink) retrieve S to the ER and ERGIC by retrograde trafficking (steps 4-6). S not retrieved by COPI is exported to the PM (step 5’) via the furin-containing trans-Golgi by anterograde trafficking. Middle panel: The coatomer is a hetero-heptamer of seven distinct gene products. N-terminal WD40 domains on α and β‘ subunits form the binding site for client proteins such as S. The illustration in panel A was created using Microsoft PowerPoint. Lower panel: Alignment of S residues in the extra-membrane domain of the cytosolic tail (1255-1273) showing the dibasic motif, extended motif, and clientizing substitutions at the C-terminus. B Multiple mutational hotspots in the N-terminal two-thirds of the full-length SARS-CoV-2 S, which corresponds to the ectodomain. C Few mutations occur in the S tail. D Clientized GST-S tail fusion protein pulls down ~17-fold more coatomer than wild-type S tail-GST fusion. This represents one of three biologically independent samples. E Immunofluorescence imaging of full-length wild-type and clientized S in permeabilized and non-permeabilized HeLa cells. Wild-type S localizes to the PM and early secretory compartments. Clientized S protein is absent from the PM and restricted to early secretory compartments. White outline, intra- and inter-cellular space devoid of S; white arrowheads, early secretory compartments. This represents one of two biologically independent samples. F Western blots of HeLa cells expressing wild-type or clientized full-length S. Wild-type S yields intact, unproteolyzed fractions from early secretory compartments and proteolyzed, furin-cleaved fragments from PM (left). Clientized S yields only an intact fraction from early secretory compartments (right). S0: intact S; S2, S2’: proteolyzed fragments. This represents one of two biologically independent samples.
