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

Abstract

During encystationGiardia trophozoites secrete a fibrillar extracellular matrix of glycans and cyst wall proteins on the cell surface. The cyst wall material is accumulated in encystation-specific vesicles (ESVs), specialized Golgi-like compartments generated de novo, after export from the endoplasmic reticulum (ER) and before secretion. These large post-ER vesicles neither have the morphological characteristics of Golgi cisternae nor sorting functions, but may represent an evolutionary early form of the Golgi-like maturation compartment. Because little is known about the genesis and maturation of ESVs, we used a limited proteomics approach to discover novel proteins that are specific for developing ESVs or associated peripherally with these organelles. Unexpectedly, we identified cytoplasmic and luminal factors of the ER quality control system on two-dimensional electrophoresis gels, i.e. several proteasome subunits and HSP70-BiP. We show that BiP is exported to ESVs and retrieved via its C-terminal KDEL signal from ESVs. In contrast, cytoplasmic proteasome complexes undergo a developmentally regulated re-localization to ESVs during encystation. This suggests that maturation of bulk exported cyst wall material in the Golgi-like ESVs involves both continuous activity of ER-associated quality control mechanisms and retrograde Golgi to ER transport.

Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation

“…In PP2Aas cells CWP expression, the earliest step in encystation, is inhibited. Therefore, all subsequent steps including formation of the ESV and the cyst wall are also greatly decreased [4,45,46]. Since we have never seen gPP2A-C in the nuclei, we do not know whether it is directly needed for CWP synthesis or affects a transcription factor or other upstream protein or event.…”

Proteomic analysis of Giardia: Studies from the pre- and post-genomic era

“…Using 2D-PAGE, 16 separate proteins were identified from the ESV/Golgi fraction derived from encysting trophozoites. Proteins found included several alpha giardins, heat shock proteins, metabolic enzymes, components of the 20S proteasome and the trophozoite antigens, GTA-1 and GTA-2 (Stefanic, et al, 2006). Neither CWP1 nor CWP2 were identified on the 2D-PAGE gel, however, their presence was confirmed using western blots analysis of ESV protein separated using SDS-PAGE.…”

“…Studies have involved the encystation specific vesicles (ESVs) (Luján, et al, 1995, Stefanic, et al, 2006, the ribosomes (Shirakura, et al, 2001) and the 20S proteasome (Emmerlich, et al, 1999). The ESVs of G. duodenalis trophozoites are produced after induction of encystation and contain the cyst wall proteins (CWP1, CWP2) as well as other proteins (Luján, et al, 1995).…”

Encystation commitment inGiardia duodenalis: a long and winding road

“…Parasite, 2009, 16, 247-258 tion proceeds (Stefanic et al, 2006). This maturation involves three major processes: .…”

“…. Quality control of cargo Misfolded cargo molecules are most likely processed by proteasome and Sec61 translocon activities to allow ESV-to-ER retrograde transport of proteins within COPIcoated vesicles where chaperons such as BiP cycle between these compartments (Marti et al, 2003;Stefanic et al, 2006). .…”