A Nanobody Collection for the Identification of Client Binding Sites on the Sorting Adaptor Vps74p

In the Golgi apparatus, glycoproteins are processed through sequential modifications by Golgi-resident glycosyltransferases, which are distributed across different cisternae. To prevent the mislocalization of these enzymes during cisternal maturation, the yeast peripheral membrane protein Vps74p acts as a sorting adaptor. It recruits glycosyltransferases into COPI-coated vesicles for retrograde transport. This mechanism is evolutionarily conserved. Notably, the human homolog of Vps74p, GOLPH3, is a recognized oncogene. Amplification of GOLPH3 is associated with poor prognosis in several cancers. Therefore, understanding how Vps74p-family proteins sort their clients provides insight into Golgi organization and may reveal potential targets for novel cancer therapeutics. However, the precise client binding sites on Vps74p have remained elusive. In this study, I generated a collection of nanobodies against Vps74p to map these binding sites. Anti-Vps74p nanobodies were selected from a synthetic yeast surface-display library. A subsequent functional screen identified inhibitory nanobodies that produced dominant-negative effects in yeast cells. One such nanobody, Vps74InNb#4, which cross-reacts with GOLPH3, disrupted the recruitment of Vps74p to the Golgi in cells and reduced its client binding in vitro. Using AlphaFold3, I mapped the Vps74InNb#4 epitope to an evolutionarily conserved loop. By analyzing competitive binding between the nanobody, Sac1p, Arf1p, and client proteins for Vps74p, I determined that the client-binding site is a pocket formed by two loops. This conclusion was confirmed by site-directed mutagenesis of these loops, guided by AlphaFold2 predictions. Furthermore, I demonstrated that the PI(4)P-binding site and an adjacent beta-hairpin structure are integral components of the client-binding pocket. These findings were successfully extended to the human counterpart, GOLPH3, indicating that the Vps74InNb#4 nanobody discovered here shows potential for developing GOLPH3-targeted cancer therapies.