真核膜囊泡包含特定的蛋白质集合，决定了囊泡的功能和与特定目的的穿梭。贾第鞭毛虫含有未知的细胞质囊泡，与人髓性白血病因子（MLF）的同源物命名为MLF囊泡（MLFVs）相关。先前的研究表明，MLF也和两个自噬机器FYVE和ATG8样蛋白共定位，而MLFVs是应对siRNA、MG132和氯喹等药物处理时蛋白酶体或自噬底物的应激诱导物。使用细胞周期依赖性激酶2的突变蛋白CDK2m3，以了解异常蛋白是否被定位到降解复合体。有趣的是，CDK2m3上调了MLF，它们共定位于同一囊泡中。自噬是一种自我消化过程，激活以去除受损蛋白质，以预防在应对各种压力中的细胞死亡。由于贾第鞭毛虫缺乏某些自噬机器，自噬机制在其中的作用还不清楚。在本研究中，我们在哺乳动物细胞中测试了六种自噬体和应激诱导物，包括MG132、雷帕霉素、氯喹、诺可唑、DTT和G418，并发现治疗会增加贾第鞭毛虫中反应性氧化物的产生和囊泡数量和MLF、FYVE和ATG8样蛋白的水平。五种应激诱导物也提高了CDK2m3蛋白水平和囊泡数量。使用应激诱导剂和MLF敲低系统，我们确定了CDK2m3应激诱导正被MLF调节。一个降低自噬体的药物3-甲基腺嘌呤，能减少MLF和CDK2m3囊泡和蛋白质。此外，使用CRISPR/Cas9系统敲低MLF，减少了在应激诱导剂处理时的细胞存活率。我们新开发的CRISPR/Cas9补充系统表明，MLF的补充能够在应对应激诱导剂时恢复细胞存活。此外，人类MLF2，像贾第鞭毛虫MLF一样，可以增加贾第鞭毛虫中囊泡蛋白的表达和囊泡形成，并能共定位于MLFVs并与MLF相互作用。我们的研究结果表明，MLF家族蛋白质在进化上是功能上保守的。我们的研究结果还提示MLF在应对压力条件下的存活中具有重要作用，而MLFVs与自噬囊泡具有相似的应激诱导特性。©2023年作者。

The eukaryotic membrane vesicles contain specific sets of proteins that determine vesicle function and shuttle with specific destination. Giardia lamblia contains unknown cytosolic vesicles that are related to the identification of a homolog of human myeloid leukemia factor (MLF) named MLF vesicles (MLFVs). Previous studies suggest that MLF also colocalized with two autophagy machineries, FYVE and ATG8-like protein, and that MLFVs are stress-induced compartments for substrates of the proteasome or autophagy in response to rapamycin, MG132, and chloroquine treatment. A mutant protein of cyclin-dependent kinase 2, CDK2m3, was used to understand whether the aberrant proteins are targeted to degradative compratments. Interestingly, MLF was upregulated by CDK2m3 and they both colocalized within the same vesicles. Autophagy is a self-digestion process that is activated to remove damaged proteins for preventing cell death in response to various stresses. Because of the absence of some autophagy machineries, the mechanism of autophagy is unclear in G. lamblia.In this study, we tested the six autophagosome and stress inducers in mammalian cells, including MG132, rapamycin, chloroquine, nocodazole, DTT, and G418, and found that their treatment increased reactive oxygen species production and vesicle number and level of MLF, FYVE, and ATG8-like protein in G. lamblia. Five stress inducers also increased the CDK2m3 protein levels and vesicles. Using stress inducers and knockdown system for MLF, we identified that stress induction of CDK2m3 was positively regulated by MLF. An autophagosome-reducing agent, 3-methyl adenine, can reduce MLF and CDK2m3 vesicles and proteins. In addition, knockdown of MLF with CRISPR/Cas9 system reduced cell survival upon treatment with stress inducers. Our newly developed complementation system for CRISPR/Cas9 indicated that complementation of MLF restored cell survival in response to stress inducers. Furthermore, human MLF2, like Giardia MLF, can increase cyst wall protein expression and cyst formation in G. lamblia, and it can colocalize with MLFVs and interact with MLF.Our results suggest that MLF family proteins are functionally conserved in evolution. Our results also suggest an important role of MLF in survival in stress conditions and that MLFVs share similar stress-induced characteristics with autophagy compartments.© 2023. The Author(s).