Studies On Lipid Transfer Activity Of PDZD8 And Its Cellular Function At ER-late Endosome/Lysosomes Membrane Contact Sites

Backgroud: Membrane contact sites between endoplasmic reticulum(ER)and late endosomes/lysosomes(LE/lys)are emerging as critical hubs for diverse cellular events(endosomal maturation and transport,maintenance of intracellular lipid and ion homeostasis),and changes in their extents are linked to severe neurological diseases.However,molecular composition,regulation and coordination of tether protein complexes between these membrane contacts are still elusive.Objective: To identify key factors at ER-LE/lys MCSs tether protein,and further elucidate the roles and mechanisms of the key factor at ER-LE/lys MCSs,and eventually explore the cellular functions of the ER-LE/lys MCSs mediated by the key factor.Methods: To identify unknown proteins that may interact with Protrudin,a known ER-LE/lys MCSs tether protein,we performed GFP-trap assays followed by mass spectrometry(MS)analysis.A SMP domain containing protein named PDZ domain containing protein 8(PDZD8)strongly interested us due to its potential lipid transfer activities.Therefore we explored cellular localizations of PDZD8 and Protrudin by live-cell confocal microscopy.We further examined the localization of PDZD8 relative to LE/lys,ER and mitochondria by super-resolution Lattice-SIM microscopy.Based on protein domain analysis,we constructed multiple truncated mutants of PDZD8,and confirmed the regions of PDZD8 targeting ER and LE/lys,respectively,by live-cell confocal microscopy.We identified PDZD8-interacting proteins by GFP-trap assays followed by MS analysis,and found that PDZD8-C1-CC region targeted LE/lys by directly binding Rab7.We next tested the possibility that PDZD8 might directly bind and transport lipids.We confirmed the lipid binding ability of PDZD8 by mass spectrometry(MS)analysis and lipid binding assay in vitro.We next employed a fluorescence resonance energy transfer(FRET)–based lipid transfer assay to examine whether the SMP domain can transfer lipids between membranes in vitro.We further investigated whether PDZD8 played important roles in the process of LE/lys trafficking and neurite outgrowth using Neuronal growth factor(NGF)stimulated PC12 differentiation PC12 cells by confocal microscopy.Results: We showed that PDZD8 was a tether at ER-LE/lys MCSs and interacted with the TM domain of Protrudin at such MCSs.PDZD8 anchored into the ER membrane with its N terminal TM domain while the C-terminal C1-CC region recognized LE/lys membrane through directly binding to Rab7.PDZD8 mediated ER-LE/lys MCSs were regulated by Rab7 activities.The SMP domain bound to glycerophospholipids and ceramides and mediate lipids transport between membranes in vitro.Functionally,PDZD8 mediated proper cellular positioning of LE/lys and promoted neurite outgrowth,which was lipid transfer activity dependent.Conclusion: PDZD8 mediated ER-LE/lys MCSs and its SMP domain bound to glycerophospholipids and ceramides and mediate lipids transport between membranes in vitro.PDZD8 functions as a tether at ER-LE/lys MCSs and is required for LE/lys positioning and neurite extensions through lipid transfer activities mediated by SMP domain.