Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway

To survive in the environment and infect a new host, Giardia lamblia secret es an extracellular cyst wall using a poorly understood pathway. The two cy st wall proteins (CWPs) form disulphide-bonded heterodimers and are exporte d via novel encystation-specific secretory vesicles (ESVs). Exposure of euk aryotic cells to dithiothreitol (DTT) blocks the formation of disulphide bo nds in nascent proteins that accumulate in the endoplasmic reticulum (ER) a nd induces an unfolded protein response (UPR). Proteins that have exited th e ER are not susceptible. Exposure to DTT inhibits ESV formation by > 85%. Addition of DTT to encysting cells causes rapid (t(1/2) < 10 min), reversib le disappearance of ESVs, correlated with reduction of CWPs to monomers and reformation of CWP oligomers upon removal of DTT. Neither CWPs nor ESVs ar e affected by mercaptoethanesulphonic acid, a strong reducing agent that do es not penetrate cells. DTT does not inhibit the overall protein secretory pathway, and recovery does not require new protein synthesis. We found evid ence of protein disulphide isomerases in the ESV and the surface of encysti ng cells, in which they may catalyse initial CWP folding and recovery from DTT. This is the first suggestion of non-CWP proteins in ESVs and of enzyme s on the giardial surface. DTT treatment did not stimulate a UPR, suggestin g that Giardia may have diverged before the advent of this conserved form o f ER quality control.