Adaptor Protein Complex 1 Mediates the Transport of Lysosomal Proteins from a Golgi-like Organelle to Peripheral Vacuoles in the Primitive EukaryoteGiardia lamblia

Abstract

Giardia lamblia is an early branching protist that possesses peripheral vacuoles (PVs) with characteristics of lysosome-like organelles, located underneath the plasma membrane. In more evolved cells, lysosomal protein trafficking is achieved by cargo recognition involving adaptor protein (AP) complexes that recognize specific amino acid sequences (tyrosine and/or dileucine motifs) within the cytoplasmic tail of membrane proteins. Previously, we reported that Giardia has a tyrosine-based sorting system, which mediates the targeting of a membrane-associated cysteine protease (encystation-specific cysteine protease, ESCP) to the PVs. Here, we show that Giardia AP1 mediates the transport of ESCP and the soluble acid phosphatase (AcPh) to the PVs. By using the yeast two-hybrid assay we found that the ESCP tyrosine-based motif interacts specifically with the medium subunit of AP1 (Gimicroa). Hemagglutinin-tagged Gimicroa colocalizes with ESCP and AcPh and coimmunoprecipitates with clathrin, suggesting that protein trafficking toward the PVs is clathrin-adaptin dependent. Targeted disruption of Gimicroa results in mislocalization of ESCP and AcPh but not of variant-specific surface proteins. Our results suggest that, unlike mammalian cells, only AP1 is involved in anterograde protein trafficking to the PVs in Giardia. Moreover, even though Giardia trophozoites lack a morphologically discernible Golgi apparatus, the presence of a clathrin-adaptor system suggests that this parasite possess a primitive secretory organelle capable of sorting proteins similar to that of more evolved cells.

Characterization of SNAREs Determines the Absence of a Typical Golgi Apparatus in the Ancient Eukaryote Giardia lamblia

“…To test whether the bioinformatic data correctly predict the compartment or pathway of a given SNARE, the 17 coding genes for putative gSNAREs were amplified by PCR from G. lamblia genomic DNA, cloned into an HA fusion vector, and transfected into G. lamblia trophozoites (37). It was shown previously that small tags such as the HA epitope do not influence the behavior of Giardia proteins and do not affect their subcellular localization (4,20). The HA epitope was inserted at the N terminus of these constructs because SNARE proteins do not have an N-terminal signal peptide (31) and also because it has been shown that this procedure accurately reflects the localization of SNARE proteins in other eukaryotic cells (54).…”

The Unique Endosomal/Lysosomal System of Giardia lamblia

“…Several groups have presented evidence for a role of clathrin in endocytosis in Giardia (Touz et al, 2004;Hernandez et al, 2007;Gaechter et al, 2008;Rivero et al, 2010). However, neither typical membrane-associated clathrin lattices nor emerging clathrin-coated pits have been observed in this parasite.…”

“…In mammalian cells, it has been shown that TCs contain mannose-phosphate receptors, clathrin, Golgi-localizing Gamma-ear containing ARF-binding proteins (GGAs), and/or adaptor protein 1 (AP-1), and it was suggested that these might be uncoated during the TC-endosome fusion or could become integrated into the endosome membrane (Polishchuk et al, 2006). Possibly supporting this hypothesis, giardial clathrin and AP-1 were observed not only on ER-exist sites but also in PVs (Marti et al, 2003a;Marti et al, 2003b;Touz et al, 2004;Gaechter et al, 2008). As we said, the ER tubular-vesicular network apparently extends to and contacts the PVs in the periphery of the cell (Abodeely et al, 2009b) but it was recently reported that no ER membranes invade the space occupied by PVs (Faso and Hehl, 2011).…”

“…The regulated pathway takes place only during encystation and associates with the appearance of encystation-specific vesicles (ESVs), which transport cyst wall components to the plasma membrane of the encysting cell and release their content to the cell exterior during cyst wall formation (Reiner et al, 1989;McCaffery and Gillin, 1994;Lujan et al, 1995;Sun et al, 2003). A selective pathway sorting proteins to the endosomal/lysosome membrane system has been recently demonstrated by our group, although Giardia do not possess distinctive endosomes or lysosomes Touz et al, 2004;Rivero et al, 2010;Rivero et al, 2011).…”

“…In contrast to most eukaryotes, Giardia has highly polarized vacuoles, located underneath the plasma membrane of the dorsal side, which combine some of the characteristics of endosomes and some of lysosomes (Lindmark, 1988;Lanfredi-Rangel et al, 1998;Touz et al, 2004). These PVs, distinguished by their localization, are about 150 nm in diameter with variable oval shapes and contain a core of low electron density (Figure 3).…”