Fig. 10

MLFV compartments are induced by six autophagosome-inducing agents, but decreased by 3-MA, an autophagosome-reducing agent. MLF interacts with FVYE and ATG8L in their compartments. MLFVs are maintained at a basal level in vegetative trophozoites but increases during encystation. During encystation, unwanted trophozoite specific proteins are accumulated. Accumulation of toxic undegraded proteins may induce protein clearance pathway to promote cell survival during encystation. Therefore, MLF, FVYE, and ATG8L, are induced for cooperation in protein clearance during encystation. BIP, as a chaperone, binds to unfolded proteins and helps refold the proteins to a soluble form. Unfolded proteins can also be degraded by ubiquitin–proteasome systems. In the presence of stress inducers, excess of misfolded proteins may overwhelm the capacity of chaperone and proteasome systems and lead to toxicity to cells. The aberrant protein, CDK2m3, may be degraded by specific clearance pathway through entering MLFV compartments. The compartments may fuse with lysosomes (peripheral vesicles, PV) to recycle materials and control energy balance. Treatment with chloroquine or nocodazole, an inhibitor of fusion of the compartments and lysosome, may impair the process and lead to accumulation of aberrant proteins, such as CDK2m3. Treatment with MG132, a proteasome inhibitor, may impair protein quality control, leading to unfolded protein response (UPR). Treatment with rapamycin, an autophagy inducer, may activate UPR. Treatment with DTT, an ER stress inducer, may activate UPR. Treatment with G418, an aminoglycoside antibiotic that interferes folding of proteins, may generate UPR. UPR causes accumulation of aberrant proteins, such as CDK2m3. All the six agents that promote autophagosome accumulation in higher eukaryotes, can induce MLF protein and vesicles in G. lamblia. Five can induce CDK2m3 protein and vesicles. 3-MA, an autophagosome-reducing agent in higher eukaryotes, can reduce CDK2m3 and MLF proteins and vesicles in G. lamblia. Our findings suggest that MLFVs involve in the clearance of CDK2m3 during stresses and share similar characteristics with autophagosomes