Mechanistic Investigations Of Celluar Localizations And Functions Of Mammalian VPS13D

Backgroud:Human VPS13 family contained four members,namely,VPS13A,B,C,and D.VPS13A/C were reportedly lipid transporters at endoplasmic reticulum(ER)-mitochondria/lipid droplets(LDs)(VPS13A)or ER-late endosomes/LDs(VPS13C)membrane contact sites(MCSs),while VPS13B was suggested to be a Golgi apparatus-associated protein required for Golgi integrity and neurite outgrowth.VPS13 proteins are of great biomedical interest as loss-of-function mutations in human VPS13 proteins were associated with genetic diseases.Loss-of-function mutations of VPS13D caused a type of autosomal recessive ataxia with abnormal mitochondrial morphology,reduced energy generation,and lipidosis.However,the cellular localization and functions of VPS13D are largely unknown.Objective:To explore the localization and celluar functions of mammalian VPS13D.Methods:We ectopically expressed a superfolder green fluorescent protein(sf GFP)-tagged VPS13D(transcript variant 2,NM?018156)(VPS13D^sf GFP),in which VPS13D was internally tagged with sf GFP to preserve its cellular localization and functions.Meanwhile,we explored the localization of endogenous VPS13D by immunofluorescence staining(IF)after pre-clearing the antibody.With the utilization of laser confocal microscopy as well as high-resolution airyscan live cell imaging(LSM900,Zeiss)and Leica SP8 equipped with lightning super-resolution module,we examined the localizations of VPS13D upon complete medium(CM)or starvation treatment,in which cells were initially loaded with oleic acid(OA)in CM and then were changed to EBSS(OA/EBSS).In addition,we examined the localizations of VPS13D truncated mutants including VPS13?C,VPS13D?N and DUF1162 via confocal imaging and cell fractionation.Then,we found that VPS13D and its truncated mutation DUF1162 interacted with the endosomal sorting complex required for transport(ESCRT)protein TSG101 by GFP-Trap.We examined the effects of RNAi-mediated VPS13D suppression on mitochondria-LD MCSs and ER-mitochondria MCSs using quantitative MCSs reporter cell lines.We also examined the roles of VPS13D in mitochondrial functions including the synthesis of mitochondria DNA and mitochondrial Ca²⁺uptake by confocal microscopy.We also explored the VPS13D-interacting proteins by GFP-Trap assays.Results:VPS13D was mainly cytosolic with a significant fraction on the outer mitochondrial membrane(OMM)and a small portion on LDs upon CM treatment.We found the binding of VPS13D to LDs was mediated through the two amphipathic helices in the VPS13?C region.A small fraction of VPS13D?N was localized to mitochondria in the absence of OA,while its recruitment to OMM was enhanced upon OA stimulation.The DUF1162 domain of VPS13D interacts with ESCRT-I protein tumor susceptibility gene101(TSG101)and is indispensable for TSG101 recruitment to LDs.VPS13D is required for the maintenance of mitochondria-LD MCSs.Intriguingly,VPS13D suppression markedly enhanced the ER-mitochondria interactions.Moreover,we found that interactions between ER and other organelles,including Golgi apparatus,peroxisomes,early endosomes,and late endosomes/lysosomes(LE/lys)were not substantially altered upon VPS13D suppression.We found that replicating mt DNA localized in mitochondria with a density of?0.28?m^-1in control cells,with some preferentially localized at tips of mitochondria.In contrast,VPS13D suppression reduced the density of replicating mt DNA by a factor of 4,and strongly increased the size of replicating mt DNA.Suppression of dynamin-related protein 1(Drp1)or mitochondrial fission factor(Mff)significantly decreased the number of replicating mt DNA and caused aggregation of replicating mt DNA,while suppression of mitofusion2 also altered the density or size of replicating mt DNA but to a less extent than Drp1 or Mff inhibition.Contrary to control cells in which ionomycin-induced mitochondrial calcium influx was transient,VPS13D suppression led to elongated mitochondrial Ca²⁺uptake upon ionomycin stimulation.We found that VPS13D interacted with VAPB,and AAA-ATPase VCP/P97.VAPB-PTPIP51 suppression significantly rescued ER-mitochondria hypercoupling at both periphery and peri-nuclear regions in VPS13D-suppressed cells.Inhibition of VCP/P97 by its allosteric inhibitor NMS873 caused ER-mitochondria hypercoupling,resembling the phenotype induced by VPS13D suppression.Conclutions:Our findings suggested VPS13D as a mitochondrial/LDs protein,which was enriched at mitochondria-LD MCSs under starvation.Functionally,VPS13D suppression resulted in the reduction of LDs-mintochondrial MCSs,intensive endoplasmic reticulum-mitochondria MCSs and defects of mitochondrial functions.Mechanistically,VPS13D coupled with VCP/P97 to negatively regulate interaction of ER-mitochondria MCSs.