Extended Data Fig. 3: PVC payloads Pdp1 and Pnf are loaded via N-terminal packaging domains.

a, Workflow for studying payload loading in PVCs. Only fully assembled PVC particles (along with any loaded payloads) localized to the pellet after ultracentrifugation; low molecular weight free proteins, however, did not pellet. This enabled the identification of loaded payloads via denaturing western blot of the pellet fraction. b, The PVC payload Pdp1 contains a predicted N-terminal packaging domain. The N-terminal domain (NTD) of Pdp1 is likely disordered as it exhibits a low pLDDT score in AlphaFold (it appears as a long disordered extension in the protein’s predicted structure) as well as a high PONDR VSL2 score⁴⁹. This N-terminal disordered region could serve as a “packaging domain”—a molecular identifier to assist the PVC’s loading machinery in identifying and loading the proper payloads. c-d, N-terminal packaging domains of Pdp1 (c) and Pnf (d) are necessary and sufficient for loading of these proteins into a PVC particle. Pdp1 and Pnf were tagged with C-terminal HiBiT tags (to enable detection via western blot) and were serially truncated to locate minimal sequences capable of loading these payloads into a PVC particle. Domains on the N termini of each payload protein were sufficient for loading, and truncation of these NTDs inhibited loading, indicating the NTDs of Pdp1 and Pnf serve as packaging domains for this PVC. As in Fig. 1e, the baseplate protein (Pvc12) was chosen as a loading control for this western blot as an equivalent number of Pvc12 units are found in each PVC particle. Note that in panel (d), while both the Pvc12 and Pnf bands were visualized on the same gel, two different exposure times were used (to avoid oversaturation of the brighter Pvc12 bands).