The Retromer complex in the primitive parasite Giardia lamblia

MIRAS S; RIVERO MR; ZAMPONI N; ROPOLO AS; TOUZ MC

Encuentro; XXIV Reunión Científica Anual. Sociedad de Protozoología. SAP; 2010

Retromer is an evolutionary conserved multi-subunit complex that mediates retrograde transport of transmembrane proteins from endosomes to the trans-Golgi network (TGN). The core of the retromer protein complex is associated with the cytosolic face of endosomes and involves a large subcomplex comprised of Vps35, Vps29, and Vps26 (Vacuolar protein sorting) along with a smaller subcomplex formed by SNX1 and SNX2 (Sorting Nexins). The best characterized transmembrane proteins sorted by retromer are acid hydrolase receptors like the vacuolar protein sorting 10 (Vps10) in the yeast Saccharomyces cerevisiae and the mannose 6-phosphate receptors (MPR) in mammals. Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells including an internal membrane system, cytoskeleton, and secretory pathways. However, because Giardia does not contain a recognized Golgi apparatus or a typical endosome/lysosome system, it is possible that this complex shows many particularities. By searching the GiardiaDB, we found genes that codify to the subcomplex gVps35, gVps29, and gVps26 but not SNX1 or SNX2. Bioinformatic tools showed that these proteins exhibit structural conservation with those of mammalian and yeast. Interestingly, a conserved motif important for Vps35-Vps26 interaction was observed the Vps35 Giardia’s homologue. IFA and confocal microscopy of stably transfected trophozoites over-expressing gVps-HA fusion proteins, showed that the gVps localize mainly around the nuclei partially colocalizing with the endoplasmic reticulum marker BIP (Immunoglobulin Binding Protein). In addition, gVps35 and gVps 29 but not gVps26 also colocalized with a lysosomal-like peripheral vacuoles marker. By yeast-two hybrid assays, we found that gVps35 directly binds to Giardia hydrolase receptor (gHR) and the gVps29 suggesting that the retrieval of the hydrolase receptor is in charge of a retromer complex.  The high degree of conservation of retromer implies an early evolutionary appearance and functional indispensability. These results will greatly increase our understanding of unique aspects of lysosomal trafficking in Giardia and also shed light on the mechanisms of protein targeting that have been acting over hundreds of millions of years of eukaryotic evolution.