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 secretes an extracellular cyst wall using a poorly understood pathway. The two cyst wall proteins (CWPs) form disulphide-bonded heterodimers and are exported via novel encystation-specific secretory vesicles (ESVs). Exposure of eukaryotic cells to dithiothreitol (DTT) blocks the formation of disulphide bonds in nascent proteins that accumulate in the endoplasmic reticulum (ER) and induces an unfolded protein response (UPR). Proteins that have exited the ER are not susceptible. Exposure to DTT inhibits ESV formation by > 85%. Addition of DTT to encysting cells causes rapid (t1/2 < 10 min), reversible disappearance of ESVs, correlated with reduction of CWPs to monomers and reformation of CWP oligomers upon removal of DTT. Neither CWPs nor ESVs are affected by mercaptoethanesulphonic acid, a strong reducing agent that does not penetrate cells. DTT does not inhibit the overall protein secretory pathway, and recovery does not require new protein synthesis. We found evidence of protein disulphide isomerases in the ESV and the surface of encysting 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 enzymes on the giardial surface. DTT treatment did not stimulate a UPR, suggesting that Giardia may have diverged before the advent of this conserved form of ER quality control.
Reiner
D S
DS
Department of Pathology, Division of Infectious Diseases, University of California San Diego, School of Medicine, San Diego, CA 92103-8416, USA.
McCaffery
J M
JM
Gillin
F D
FD
D000818:Animals; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D016829:Giardia lamblia / Q000187:drug effects / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D015800:Protozoan Proteins / Q000187:drug effects / Q000378:metabolism*; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*
D015800:Protozoan Proteins; D019704:Protein Disulfide-Isomerases; D004229:Dithiothreitol