Mechanism Of Endocytic Trafficking Mediated By SNX-BARs

Endosomes are key platforms for transmembrane receptor and lipid sorting,as well as for cell signaling.Cell surface receptors arrive from endocytic or anterograde trafficking routes,and are either sent to the lysosome for degradation,or recycled to other compartments,including the plasma membrane and the trans-Golgi network(TGN).Endosomal trafficking is indispensable for maintaining plasma membrane homeostasis,and is tightly controlled by many pivotal protein machineries.It should be noted that the endosomal trafficking is also dynamically regulated by outer cell stress.Consequently,dysregulation of this process contributes to the development of a variety of human diseases,including Parkinson’s disease,Alzheimer’s disease,and cancer.Finally,many intracellular pathogens,including vacuolar bacteria,human papilloma virus(HPV),and SARS-CoV-2,hijack the endosomal trafficking pathways for their infection and replication.Key protein machineries important for the sequence-dependent recycling include the retriever complex(VPS26C-VPS35L-VPS29),the WASH complex,TBC1D5 and the evolutionarily conserved retromer complex(VPS35/VPS26/VPS29 in higher eukaryotes)and members of the Sorting Nexin family(SNXs).A subset of SNX proteins possessing a Bin/Amphiphysin/Rvs(BAR)domain,in addition to the phoxhomology(PX)domain,have been linked with the retromer complex.The retromerrelated SNX-BAR proteins(referred as SNX-BARs herein)SNX1,SNX2,SNX5,SNX6,and SNX32 form heterodimeric complexes and are critical for both endosometo-plasma membrane recycling and endosome-to-TGN retrieval via promoting membrane tabulation or a yet-to-be determined mechanism.Due to the specific expression of SNX32 in neuron,we do not include much discussion about it.Current models suggest that SNX-BARs promote the endosome-to-plasma membrane recycling via associating with SNX27 and retromer,with the PDZ domain of SNX27 as the predominant cargo-recognition module.For endosome-to-TGN trafficking,one of the best-characterized cargoes is cation-independent mannose 6-phosphate receptor(CI-MPR),which is necessary to deliver newly synthesized lysosomal hydrolases to the endosomal lumen and thus critical for lysosomal function,and thus being critical for the maintainance of lysosomes.However,previous studies have provided contradictory models regarding the role of retromer and SNX-BARs in the endosometo-TGN retrieval of CI-MPR.Work from many different labs has initially supported the idea that retromer is necessary for the retrieval of CI-MPR,likely through a direct association with its cytoplasmic tail,in particular,the hydrophobic WLM motif.However,recent work by Cullen and Steinberg has provided a very different model,in which SNX-BARs,but not retromer,associate with the cytoplasmic tail of CI-MPR and mediate its trafficking.Interestingly,affinity-based mass spectrometry and protein trafficking experiments showed that SNX-BARs are also responsible for the transport of IGF1R and SEMA4C.The importance of endosomal sorting pathways for cellular homeostasis and functions is further highlighted by the findings that many viral and bacterial pathogens target these pathways for survival and growth.For instance,whereas retromer activity restricts intracellular growth of the bacteria Chlamydia,Chlamydia counteracts the host activity through a direct binding between its effector protein IncE and SNX5/SNX6,suggesting that although mammalian retromer does not bind stably to SNX-BARs,these two complexes are functionally related.Ectopic IncE production disrupts CI-MPR endosomal trafficking,likely by competing with CI-MPR for SNX5 binding.However,it remains poorly understood how SNX-BARs selectively bind to CI-MPR or other cargoes,including IGF1R and SEMA4C.Using GST pull-down and ITC assays,we identified the SNX5 or SNX6 from SNX-BARs as the critical component to bind to the 164 amino acids within the cytoplasmic tail of CI-MPR.Next,we mapped the necessary region for the interaction between SNX5 and CI-MPR via GST pull-down and ITC assays.Since many labs consider SNX3-retromer is involved in the retrieval of CI-MPR,we tested the binding affinity between CI-MPR and SNX3-retromer in vitro.Yet,little binding affinity,if any,could be obtained under our experimental conditions,suggesting that SNX-BARs dominant the retrieval of CI-MPR,with SNX3-retromer likely playing a supplementary role.Furthermore,we managed to determine the molecular mechanism of cargoes binding to SNX5 using NMR spectroscopy.Our biochemical data showed that that SNX5 interacts with CI-MPR through specific amino acids located within its long a helix hairpin structure.Interestingly,the long a helix hairpin structure is the key region for binding to the Chlamydia effector protein IncE.Consistently,we found that CIMPR competed with IncE for the identical binding site on SNX5.Next,we found that the proteins interacting with SNX5 share a highly conserved motif.By introducing point mutagenesis into different SNX5 binding proteins,including CI-MPR,IGF1R and SEMA4C,we verified that this conserved motif is the key region to touch SNX5.Interestingly,we found that the C-terminal of SEMA4C contained a PDZ binding motif(PDZbm)which can be recognized by the PDZ domain of SNX27.SNX27 and retromer were reported to be involved in the trafficking of SEMA4C.Interestingly,both SNX-BARs and retromer can interact with SNX27.Our surface biotinylation assays showed that SEMA4C was significantly decreased in SNX-BARs or SNX27 knockout cells,suggesting that SNX-BARs and SNX27-retromer might co-participate in the process of recognition and transport.Indeed,the introduction of alanines into the key binding residues of PDZbm and SNX5 could impaire the effective trafficking of SEMA4C.Based on the above results,we proposed a model of“SNX-BARSNX27-retromer" super-complex responsible for the retrieval of cargoes from endosomes to the plasma membrane.Since SNX3-retromer and SNX27 both recognize and transport a class of goods with conserved short motifs,is it possible for SNX-BARs to recognize and sort membrane proteins with motifs similar to CI-MPR,IGF1R and SEMA4C?Based on our biochemical experiments and sequence analysis,we came up with a conserved motif(SBM)that could be bound by SNX-BARs.The bipartite SBM consists of two hydrophobic streches linked via a short loop.Then we used bioinformaticsto screen and analyze the possible SBM-containing candidates.Finally,we obtained 71 SBMcontaining candidates including TRAILR1 and PTHR.We found that PTHR and TRAILR1 could be transported by SNX27-retromer,which is consistent with our hypothesis.Next,we introduced mutations within the SBM of TRAILR1 and PTHR,we found that the binding affinity of the two proteins with SNX-BARs were weakened.Interestingly,although TRAILR1 did not contain a PDZbm,SNX27 knockout still affected its content in plasma membrane,which further confirmed our super-complex model.After clarifying the recognition mechanism of SNX27 and SNX-BARs on TRAILR1,we further explored their effects on the biological function of TRAILR1.TRAILR1 can induce TRAIL-based apoptosis.We found that TRAIL induced apoptosis in SNX-BARs or SNX27 knockout cells was significantly lower than that in wild-type cells,suggesting the important regulatory role of SNX27 and SNX-BARs in apoptosis.In order to address the formation of super-complex,we showed that the FERM domain of SNX27 bound to the N-terminal of SNX1 through a conserved DLF motif.The crystal structure of SNX1 and SNX27 showed that SNX1 bound to a highly conserved hydrophobic region of SNX27 through the core DLF motif,and this region was surrounded by positively charged residues.Immunofluorescence assay showed that disruption of the binding would impair the retrieval of TRAILR1.By analyzing the co-localization of SNX27 and VPS35,we found that the interaction of SNX1SNX27 was also very important for the recruitment of SNX27 to endosome.These results suggest that SNX-BARs can not only promote the deformation of cell membrane,but also play an important physiological role in regulating the endocytic trafficking.In conclusion,using the methods of structural biology,biochemistry,cell biology and bioinformatics,we found a new mechanism for SNX-BARs to recognize and sort cargoes through SBM from endosomes to Golgi or plasma membrane.Next,we propose a new protein machinery "supercomplex" in the endocytic transport from endosomes to plasma membrane.Through crystal structure,we find that the supercomplex is mainly formed by the FERM domain of SNX27 and the DLF motif at the N-terminal of SNX1 or SNX2.Disrupting the binding will reduce the effective transport of some cargoes,such as TRAILR1.Our results show that the assembly of coat complexes in higher eukaryote is of great significance and the new concept and physiological function of "super-complex" may lead to a new research direction in the discipline.The significance of our research lies in:(1)Demonstrate SBM is recognized by SNX-BARs for the first time.(2)Predict 71 SBM candidates by bioinformatics.(3)Verify some SBM candidates,such as PTHR and TRAILR1.(4)Bring up a new assembly model of coat protein in endocytic trafficking.(5)The mechanism of the SNX27-SNX1 complex was analyzed by crystal structure,which guided a new research direction in the field.Our work subverts the classical model of recognition of CI-MPR by retromer in the field,indicating the difference between mammalian and yeast endosome sorting pathways,and illustrating the importance and complexity of endosomal sorting in higher organisms.