gVPS10P-like receptor: a novel protein involved in sorting of soluble hydrolases in the protozoan parasite Giardia lamblia.

MIRAS S; RIVERO MR; FELIZIANI C; ZAMPONI N; QUIROGA R; ROPOLO AS; TOUZ MC

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias.; 2011

Eukaryotic cells contain numerous distinct membrane bound organelles. To maintain the functional identities of these organelles, specific sets of proteins must be efficiently sorted and delivered to each intracellular compartment. The targeting of lysosomal enzymes from their site of synthesis in the rough endoplasmic reticulum to their final destination in lysosomes is directed by a series of protein and recognition signals. For example, in S. cerevisiaeS. cerevisiae the Carboxypeptidase Y (CPY), a soluble vacuolar hydrolase, is diverted from the secretory pathway to the trans-Golgi network (TGN), in a manner analogous to soluble lysosomal proteins that are transported to the late endosome by the mannose 6-phosphate receptor in mammalian cells. In the TGN, the vacuolar sorting receptor Vps10p interacts with CPY. The receptor/ligand complex is then packaged into vesicles and transported to the endosome en route to the vacuole. Although, lysosomal hydrolase delivery is one of the best-characterized examples of an intracellular protein-sorting process in yeast and mammalian cells, this pathway is still unidentified in G. lamblia. The purpose of this work is to show our current understanding of the structure and function of the gVps10p-like receptor in the sorting of lysosomal hydrolases to the lysosome-like peripheral vacuoles (PVs) in G. lamblia. By means of bioinformatic tools, we predicted that this receptor is a type I transmembrana protein with a WD40 motif in the luminal portion corroborating this prediction using a proteinase K assay and IFA. IFA and confocal microscopy of stably transfected trophozoites overexpressing gVps10p-HA fusion protein, showed the receptor around the nuclei colocalizing with the endoplasmic reticulum marker BIP (Immunoglobulin Binding Protein). Co-expression of gVps10p-HA with the hydrolase acid phosphatase fusion protein (gAcPh-V5), showed colocalization around nuclei and in the PVs. Besides, by yeast-two hybrid assays, we found that gVps10p not only binds to gAcPh but also to other soluble hydrolases, like the cysteine proteases of the Cathepsins B family gCP1 and gCP3. On other hand, gVps10p-YQII-HA fusion protein, lacking the cytoplasmic binding motif, showed a different subcelular distribution possibly related with changes in protein trafficking signalling. These results constitute the first evidence about the occurrence of a gVps10p-like receptor in the protozoan parasite Giardia lamblia.G. lamblia. The purpose of this work is to show our current understanding of the structure and function of the gVps10p-like receptor in the sorting of lysosomal hydrolases to the lysosome-like peripheral vacuoles (PVs) in G. lamblia. By means of bioinformatic tools, we predicted that this receptor is a type I transmembrana protein with a WD40 motif in the luminal portion corroborating this prediction using a proteinase K assay and IFA. IFA and confocal microscopy of stably transfected trophozoites overexpressing gVps10p-HA fusion protein, showed the receptor around the nuclei colocalizing with the endoplasmic reticulum marker BIP (Immunoglobulin Binding Protein). Co-expression of gVps10p-HA with the hydrolase acid phosphatase fusion protein (gAcPh-V5), showed colocalization around nuclei and in the PVs. Besides, by yeast-two hybrid assays, we found that gVps10p not only binds to gAcPh but also to other soluble hydrolases, like the cysteine proteases of the Cathepsins B family gCP1 and gCP3. On other hand, gVps10p-YQII-HA fusion protein, lacking the cytoplasmic binding motif, showed a different subcelular distribution possibly related with changes in protein trafficking signalling. These results constitute the first evidence about the occurrence of a gVps10p-like receptor in the protozoan parasite Giardia lamblia.G. lamblia. By means of bioinformatic tools, we predicted that this receptor is a type I transmembrana protein with a WD40 motif in the luminal portion corroborating this prediction using a proteinase K assay and IFA. IFA and confocal microscopy of stably transfected trophozoites overexpressing gVps10p-HA fusion protein, showed the receptor around the nuclei colocalizing with the endoplasmic reticulum marker BIP (Immunoglobulin Binding Protein). Co-expression of gVps10p-HA with the hydrolase acid phosphatase fusion protein (gAcPh-V5), showed colocalization around nuclei and in the PVs. Besides, by yeast-two hybrid assays, we found that gVps10p not only binds to gAcPh but also to other soluble hydrolases, like the cysteine proteases of the Cathepsins B family gCP1 and gCP3. On other hand, gVps10p-YQII-HA fusion protein, lacking the cytoplasmic binding motif, showed a different subcelular distribution possibly related with changes in protein trafficking signalling. These results constitute the first evidence about the occurrence of a gVps10p-like receptor in the protozoan parasite Giardia lamblia.Giardia lamblia.