The Secretory Apparatus of an Ancient Eukaryote: Protein Sorting to Separate Export Pathways Occurs Before Formation of Transient Golgi-like Compartments

Abstract

Transmission of the protozoan parasite Giardia intestinalis to vertebrate hosts presupposes the encapsulation of trophozoites into an environmentally resistant and infectious cyst form. We have previously shown that cyst wall proteins were faithfully sorted to large encystation-specific vesicles (ESVs), despite the absence of a recognizable Golgi apparatus. Here, we demonstrate that sorting to a second constitutively active pathway transporting variant-specific surface proteins (VSPs) to the surface depended on the cytoplasmic VSP tail. Moreover, pulsed endoplasmic reticulum (ER) export of chimeric reporters containing functional signals for both pathways showed that protein sorting was done at or very soon after export from the ER. Correspondingly, we found that a limited number of novel transitional ER-like structures together with small transport intermediates were generated during encystation. Colocalization of transitional ER regions and early ESVs with coat protein (COP) II and of maturing ESVs with COPI and clathrin strongly suggested that ESVs form by fusion of ER-derived vesicles and subsequently undergo maturation by retrograde transport. Together, the data supported the hypothesis that in Giardia, a primordial secretory apparatus is in operation by which proteins are sorted in the early secretory pathway, and the developmentally induced ESVs carry out at least some Golgi functions.

Organelle Proteomics Reveals Cargo Maturation Mechanisms Associated with Golgi-like Encystation Vesicles in the Early-diverged Protozoan Giardia lamblia

“…The exact mechanism of this export is still elusive, however. The available data support two nonmutually exclusive models for ESV genesis: (i) lateral segregation and concentration of cyst wall material in specialized ER subcompartments (10), and/or (ii) export of cyst wall material in COPIIcoated transport vesicles that give rise to ESVs by homotypic fusion (17). In any case, analogous to the generation of vesicular tubular clusters and cis-Golgi compartments, formation of ESVs seems to require the small GTPase Sar1p 3 and thus represents a unique form of Golgi neogenesis.…”

“…In particular, the formation and reshuffling of intra-and intermolecular disulfide bonds is essential for establishing a cyst wall, because treatment with dithiothreitol (DTT) reversibly abolished cyst wall formation and appeared to dissolve ESVs (21). Interestingly, like conventional Golgi cisternae, ESVs are sensitive to brefeldin A and associate with two Golgi markers, ␤ЈCOPI and GiYip1 (4,17). This is strong evidence for their Golgi-like nature despite the unusual morphology.…”

“…Fixation and preparation for fluorescence microscopy was done as described previously (17). Briefly, cells were washed with cold PBS and fixed with 3% formaldehyde in PBS for 40 min at 20°C, followed by a 5-min incubation with 0.1 M glycine in PBS.…”

The Unique Endosomal/Lysosomal System of Giardia lamblia

“…Analysis of GlCHC expression showed that clathrin is expressed almost equally in both stages of the parasite and is located in close association with the PVs in trophozoites, and in the ESVs in immature cysts (Marti et al, 2003a;Marti et al, 2003b;Hehl and Marti, 2004;Gaechter et al, 2008). On the basis of these observations, it was suggested that recruitment of clathrin to late ESVs could serve to disperse large ESVs into smaller transport vesicles in response to the secretion signal (see below).…”

“…The fact that both constitutive and regulated mechanisms for protein transport exist in Giardia is an example of Golgi functions, since the sorting and selection process generally occurs in the trans-Golgi network in more complex cells (Gu et al, 2001). The constitutive pathway in Giardia is represented by continuous expression and trafficking to the plasma membrane of the transmembrane-anchored variant-specific surface proteins (VSPs) (Nash, 2002;Marti et al, 2003a;Touz et al, 2005). 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).…”