The Function And Mechanism Of DENN-Domain Protein FAM45A

DENN(differentially expressed in normal and neoplastic cells)domain is an evolutionarily conserved protein module.Most known DENN domain containing proteins possess guanine exchange factor(GFF)activities toward Rab proteins and catalyze the activation of Rabs.Rab proteins are a class of small G proteins.Rabs oscillate between active GTP-bound and inactive GDP-bound states and act as molecular switches in intraceullar membrane transport.Rabs regulate various important trafficking steps such as cargo selection,vesicle mobility and membrane tethering.FAM45 A protein is a novel DENN domain protein,found in a diverse range of organisms such as amoeba,sea urchin,most insects and vertebrates.However,the function of FAM45 A has been unknown.Previous study in our laboratory showed that FAM45 A localized in late endosomes/multivesicular bodies(MVB)and lysosomes,suggesting that FAM45 A was involved in late stages of endocytic transport.In this study,we established stable FAM45 A gene silencing cell lines,with the help of which we discovered that: 1)FAM45A gene silencing resulted in clustering of EEA1-positive early endosomes and CD63-positive late endosomes to the perinuclear region;2)FAM45A silencing slowed down endocytosis-mediated degradation of EGFR,and the early-to-late endosome transition was impaired;3)FAM45A silencing decreased the secretion of exosomes.These results indicate that FAM45 A regulate the motility and homeostasis of late endosomes/MVBs.To reveal the mechanism of FAM45 A,we tested the interaction of FAM45 A and Rab protein by co-immunoprecipitation and found that FAM45 A and Rab27 b co-exist in the same protein complex,suggesting that FAM45 A regulates MVB dynamics by activating small GTPases such as Rab27 b.Objective: To study the role and mechanism of FAM45 A in intracellular vesicular transport Methods: I.Effects of FAM45 A gene silencing on vesicle transport pathway 1.Establishment of stable FAM45 A gene silencing cell lines and FAM45 A gene knockout cell lines Hela cells were infected with lentiviruses containing FAM45 A shRNA or FAM45 A CRISPR sgRNA expression cassette.Genomic integration was screened using puromycin resistance.Expression levels of FAM45 A mRNA and protein were examined by q-PCR and Western blotting respectively.2.Organelle morphology changes in FAM45 A silencing cell lines In the FAM45 A silencing cell lines,the early endosomes,the late endosomes and lysosome were labeled with EEA1 antibody,CD63 antibody and Lysotracker dye respectively and imaged.3.The degradation of EGFR in FAM45 A silencing cell lines FAM45 A gene silencing and control cell lines were starved before stimulated with EGF for 0,10,90 and 150 min,and fixed.The dynamic changes in EGFR endocytosis was examined by comparing EGFR with early endosomes(mCherry-EEA1),late endosomes(CD63)and lysosome(Lamp1)using dual-color imaging.4.The secretion of exosomes in FAM45 A silencing cell lines Exosomes of FAM45 A gene silencing and control cell lines were isolated by ExtraPEG method and analyzed by Western blotting.II.The mechanism of FAM45 A 3x FLAG-FAM45 A and Citrine-Rab proteins were co-expressed in HEK293 T cells.Co-immunoprecipitation was used to detect the interaction between FAM45 A and Rab27 b.Result: I.Effects of FAM45 A gene silencing on vesicle transport pathway 1.Stable FAM45 A knockdown cell lines and FAM45 A gene knockout cell lines were established.Both FAM45 A mRNA and protein were significantly reduced in FAM45 A knockdown cell lines.In selected FAM45 A knockout cell lines,FAM45 A mRNA was reduced to background levels.2.FAM45 A gene silencing led to EEA1 and CD63 clustering to the perinuclear region.The morphology and pH of lysosomes were largely unaffected.3.FAM45 A gene silencing resulted in slower degradation of EGFR.EGFRs were trapped in early endosomes.4.FAM45 A gene silencing decreased the secretion of exosomes,although FAM45 A was not present in exosomes.II.The mechanism of FAM45 A gene FAM45 A and Rab27 b proteins exist in the same protein complex.Conclusion: 1.FAM45 A gene silencing led to early endosomes and late endosomes clustered to the perinuclear region,suggesting that FAM45 A participates in the motility of endosomes.2.FAM45 A gene silencing resulted in the retention of EGF receptors in early endosomes,showing that FAM45 A plays an important role in the transition from early-to-late endosomes.3.Exosomes originate from the fusion of multivesicular bodies with the plasma membrane,FAM45 A knockdown decrease the secretion of exosomes,suggesting that FAM45 A acts an important role in the exosomal biogenesis mechanism.4.Co-immunoprecipitation experiments showed that FAM45 A and Rab27 b exist in the same protein complex,suggesting that FAM45 A could regulate the homeostasis of multivesicular bodies by activating Rab27 b and/or other small G proteins.In summary,we examined the function and mechanism of FAM45 A,a previously uncharacterized novel DENN domain protein.We found that FAM45 A plays an important role in the motility and homeostasis of multivesicular bodies,possibly through activating small G proteins such as Rab27 b.