DENN Domain Protein FAM45A Is A Novel Regulator Of Membrane Trafficking

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.Rab proteins are a class of small G proteins that regulate various intraceullar transport events among different membrane bound vesicles.FAM45A is a protein found in ameba,some insects and vertebrates,whose function has been unknown.Using bioinformatics prediction tools,FAM45A gene is found to be similar to DENN domain containing proteins,suggesting it might play a role in membrane trafficking.We found that both tagged and endogenous FAM45A proteins were mainly localized in late endosomes and lysosomes.Further studies showed that FAM45A was involved in the intraluminal vesicles in MVE,which indicated that FAM45A was involved in intracellular vesicle transport in the late endosomes to lysosomes.FAM45A gene silencing resulted in clustering of CD63 positive late endosomes to the perinuclear region.We also found that FAM45A silencing slowed down the degradation of EGFR in lysosomes,a classical endocytosis pathway.In summary,FAM45A is a novel regulator of intracellular vesicle transport in the late endosomes/lysosomes stage.Objective:To study the role of FAM45A in intracellular vesicular transportMethods:I.FAM45A Subcellular Localization1.FAM45A localization by fluorescent protein tagpCitrine-FAM45A fusion expression vector was constructed.Hela cells were transfected with pCitrine-FAM45A together with red-fluorescent protein tagged organelle markers,such as EEA1 and Lamp1.Confocal Laser Scanning Microscope was used to determine the localization of FAM45A in various organelles.In some experiments,Hela cells were treated with apilimod or NH₄Cl to enlarge endosomes for better observation of intraluminal vescicles.2.Endogeneous FAM45A localization by immunofluorescence.Endogeneous FAM45 were identified by an antibody to FAM45A.Hela cells were incubated with FAM45A antibody and organelle markers,followed by appropriate fluorescent secondary antibodies.Confocal laser scanning microscopy was used for visualization.In some experiments,cells were treated with apilimod or NH₄Cl to enlarge membranous organelles for better observion of intraluminal vesicles.II.Effects of FAM45A gene silencing on organelle morphology and endocytosis kinetics1.Construction of stable FAM45A gene silencing cell lineHela cells were infected with lentivirus containing FAM45 shRNA expression cassette.Genomic integration was screened using puromycin resistance.The expression level of FAM45A mRNA and protein were detected by q-PCR and western blotting respectively.2.Organelle morphology changes in FAM45A silencing cell lineOrganelle morphology was examined by immunofluorescence,fluorescent protein tagged expression or Lysotracker dyes.3.The degradation of EGFR in FAM45A silencing cell lineFAM45A gene silencing and control cell lines were starved before stimulated with EGF for 0,10,90 and 150 min respectively.The degradation of EGFR was examined by western blotting.Result:I.FAM45A LocalizationFAM45A protein was mainly localized in late endosomes and lysosomes.A large number of FAM45A was found in intraluminal vesicles of MVEs.II.Effects of FAM45A gene silencing on organelle morphology and EFGR degradation1.Successfully contructed stable FAM45A knockdown cell lines.Both FAM45A mRNA and protein were significantly reduced.2.FAM45A gene silencing led to CD63 clustering to the perinuclear region.Lamp1 positive lysosomes are enlarged.3.FAM45A gene silencing resulted in slower degradation of EGFR.Conclusion:1.FAM45A is mainly localized on late endosomes and lysosomes.FAM45A is also localized in intraluminal vesicles in MVE.2.FAM45A affects the positioning of late endosomes and lysosomes.Silencing of FAM45A slows down the degradation of EGFR,suggesting that FAM45A is a novel regulator of endocytic transport.