The giardial VPS35 retromer subunit is necessary for multimeric complex assembly and interaction with the vacuolar protein sorting receptor

Abstract

The retromer is a pentameric protein complex that mediates the retrograde transport of acid hydrolase receptors between endosomes and the trans-Golgi network and is conserved across all eukaryotes. Unlike other eukaryotes, the endomembrane system of Giardia trophozoite is simple and is composed only of the endoplasmic reticulum and peripheral vesicles (PVs), which may represent an ancient organellar system converging compartments such as early and late endosomes and lysosomes. Sorting and trafficking of membrane proteins and soluble hydrolases from the endoplasmic reticulum to the PVs has been described as specific and conserved but whether the giardial retromer participates in receptor recycling remains elusive. Homologs of the retromer Vacuolar Protein Sorting (Vps35p, Vps26p, and Vps29p) have been identified in this parasite. Cloning the GlVPS35 subunit and antisera production enabled the localization of this protein in the PVs as well as in the cytosol. Tagged expression of the subunits was used to demonstrate their association with membranes, and immunofluorescence confocal laser scanning revealed high degrees of colabeling between the retromer subunits and also with the endoplasmic reticulum and PV compartment markers. Protein-protein interaction data revealed interaction between the subunits and of GlVPS35 with the cytosolic domain of the hydrolase receptor GlVps. Altogether our data provide original information on the molecular interactions that mediate assembly of the cargo-selective retromer subcomplex and its involvement in the recycling of the acid hydrolase receptor in this parasite.

Significantly Diverged Did2/Vps46 Orthologues from the Protozoan Parasite Giardia lamblia

“…This distribution is consistent with the distribution of Vps46 at the endosome and cytoplasm in yeast cells [19]. The pattern of signal enrichment close to the cell periphery is also consistent with that observed for proteins such as Giardia adaptor protein 2 (AP2) and the retromer complex component GlVPS35; both these proteins are known to localize at the PV [17]. The localization of the GlVPS46 proteins at the PV indicates that they function at this subcellular compartment.…”

Exosome Biogenesis in the Protozoa Parasite Giardia lamblia: A Model of Reduced Interorganellar Crosstalk

“…Since Giardia lacks a morphologically discernible Golgi apparatus, the reticular and peripheral localization of GlRab11 suggest that it may play a role in ER-PV communication rather than in PV docking to the plasma membrane. The trafficking between the ER and the PVs in Giardia has been addressed, showing that vesicular trafficking was possible between these two organelles [45,87,88]. Moreover, the ER marker glucose 6-phosphatase was also observed in the PVs while the soluble lysosomal acid phosphatase, a PV marker, was found in the ER as well as in the lumen of the PVs [17,89].…”

Membrane-Associated Proteins in Giardia lamblia

“…Nevertheless, the participation of non-BAR SNX3 has also been described as mediating the recruitment of the cargo-selective retromer complex [ 29 ]. Two proteins, GL50803_24488 and GL50803_16548, described by Jana et al, 2017 [ 24 ], possess high sequence similarity to the non-BAR SNX proteins, SNX15 and SNX17, respectively [ 30 , 31 , 32 ]. The data obtained so far suggest that GL50803_24488 and GL50803_16548 participate in the recovery of membrane proteins from the PVs, where they have the capacity to bind to PI3P and the cargo subunit of the retromer to drive non-tubular transport to the ER in G. lamblia [ 30 , 31 , 32 ].…”