Calcium Contributes to Polarized Targeting of HIV Assembly Machinery by Regulating Complex Stability

[Image: see text] Polarized or precision targeting of protein complexes to their destinations is fundamental to cellular homeostasis, but the mechanism underpinning directional protein delivery is poorly understood. Here, we use the uropod targeting HIV synapse as a model system to show that the viral assembly machinery Gag is copolarized with the intracellular calcium (Ca(2+)) gradient and binds specifically with Ca(2+). Conserved glutamic/aspartic acids flanking endosomal sorting complexes required for transport binding motifs are major Ca(2+) binding sites. Deletion or mutation of these Ca(2+) binding residues resulted in altered protein trafficking phenotypes, including (i) changes in the Ca(2+)–Gag distribution relationship during uropod targeting and/or (ii) defects in homo/hetero-oligomerization with Gag. Mutation of Ca(2+) binding amino acids is associated with enhanced ubiquitination and a decline in virion release via uropod protein complex delivery. Our data that show Ca(2+)–protein binding, via the intracellular Ca(2+) gradient, represents a mechanism that regulates intracellular protein trafficking.