Sorting Nexin 9 Participates in Clathrin-mediated Endocytosis through Interactions with the Core Components

Abstract

Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the αand β2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis. Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the αand β2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis. The family name sorting nexin (SNX) 1The abbreviations used are: SNXsorting nexinSH3Src homology 3APadaptor protein complexDyn2dynamin-2GSTglutathione S-transferasePRDproline-rich domainPXphoxMALDI-TOFmatrix-assisted laser desorption ionization time-of-flight. is given to a large group of proteins that are represented throughout the eukaryotic kingdom. Sorting nexins are characterized by the presence of a subclass of the phosphoinositide-binding phox (PX) domain, and it is believed that a common function of proteins in this family is to participate in sorting processes in the cell. Several members localize to endosomal structures, and some of the SNX proteins have been shown to directly interact with transmembrane receptors to regulate their sorting in the endosomal pathway (for a review, see Ref. 1Worby C.A. Dixon J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 919-931Crossref PubMed Scopus (335) Google Scholar). The localization of SNX proteins is suggested to be determined by their PX domains, although protein-protein interactions may also contribute to the membrane specificity (2Xu Y. Hortsman H. Seet L. Wong S.H. Hong W. Nat. Cell Biol. 2001; 3: 658-666Crossref PubMed Scopus (240) Google Scholar, 3Zhong Q. Lazar C.S. Tronchere H. Sato T. Meerloo T. Yeo M. Songyang Z. Emr S.D. Gill G.N. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6767-6772Crossref PubMed Scopus (130) Google Scholar). sorting nexin Src homology 3 adaptor protein complex dynamin-2 glutathione S-transferase proline-rich domain phox matrix-assisted laser desorption ionization time-of-flight. SNX9 and its close relative SNX18 are the only members of the sorting nexin family that contains an Src homology 3 (SH3) domain. SH3 domains interact specifically with short proline-containing sequences (PXXP) and are present in a large number of proteins (4Mayer B.J. J. Cell Sci. 2001; 114: 1253-1263Crossref PubMed Google Scholar). Target sequences are often located in distinct domains, referred to as proline-rich domains (PRDs). SNX9 was originally identified as a molecule that interacted with PRDs in certain metalloprotease disintegrins (ADAMs) (5Howard L. Nelson K.K. Maciewicz R.A. Blobel C.P. J. Biol. Chem. 1999; 274: 31693-31699Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Work in Drosophila showed that SNX9 (named DSH3PX1) interacted with Dock (the fly orthologue of mammalian Nck) and Dscam (Down's syndrome cell adhesion molecule) to form a complex involved in axonal guidance in the fly (6Worby C.A. Simonson-Leff N. Clemens J.C. Kruger R.P. Muda M. Dixon J.E. J. Biol. Chem. 2001; 276: 41782-41789Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 7Worby C.A. Simonson-Leff N. Clemens J.C. Huddler Jr., D. Muda M. Dixon J.E. J. Biol. Chem. 2002; 277: 9422-9428Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In addition, SNX9 was found to interact with the clathrin-binding tyrosine kinase Ack through an SH3-PRD interaction. In mammalian cells, Ack2 interacted with the receptor for epidermal growth factor, and this interaction was stabilized by SNX9. Upon stimulation with epidermal growth factor, SNX9 became tyrosine-phosphorylated and was suggested to play a role together with Ack2 in the degradation of the epidermal growth factor receptor (8Lin Q. Lo C.G. Cerione R.A. Yang W. J. Biol. Chem. 2002; 277: 10134-10138Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In a previous report (9Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar), we identified SNX9 as one of the major proteins in the cytosol from human K562 cells that bound to the appendages of adaptor protein complex 2 (AP-2). It was therefore suggested that SNX9 in some way is involved in the process of clathrin-coated vesicle formation at the plasma membrane. Clathrin-mediated endocytosis is triggered by the assembly of AP-2 and clathrin at specific sites at the plasma membrane, mediated by interactions with phosphoinositides in the membrane (10Kirchhausen T. Annu. Rev. Cell Dev. Biol. 1999; 15: 705-732Crossref PubMed Scopus (425) Google Scholar, 11D'Hondt K. Heese-Peck A. Riezman H. Annu. Rev. Genet. 2000; 34: 255-295Crossref PubMed Scopus (105) Google Scholar, 12Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (540) Google Scholar). As the clathrin coat forms, the membrane invaginates and becomes a coated pit. Once activated in the coat, AP-2 reaches out to collect transmembrane cargo proteins by protein-protein interactions (13Collins B.M. McCoy A.J. Kent H.M. Evans P.R. Owen D.J. Cell. 2002; 109: 523-535Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). A variety of accessory factors participates in these events, many of them interacting with the appendage domains of AP-2 (14Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (426) Google Scholar). At a certain stage, the large GTPase dynamin is recruited to the coated pit and forms a collar at its base (15Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (584) Google Scholar, 16Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (356) Google Scholar). The assembly of dynamin leads to fission of the clathrin-coated vesicle from the membrane, possibly by a force generated through the hydrolysis of GTP. Although it is known that dynamin plays an essential role in the later stages of endocytosis, the mechanism of action has not yet been revealed (17Sever S. Damke H. Schmid S.L. J. Cell Biol. 2000; 150: 1137-1148Crossref PubMed Scopus (196) Google Scholar, 18Song B.D. Schmid S.L. Biochemistry. 2003; 42: 1369-1376Crossref PubMed Scopus (102) Google Scholar). In addition, it has not been established how this important molecule is targeted to the membrane. In the present study, we identify dynamin-2 (Dyn2) as a major partner for SNX9 in the cytosol from hematopoietic cells and show that SNX9 assembles with Dyn2 and AP-2 at the plasma membrane. The results suggest that SNX9 plays a role in the clathrin-dependent endocytic machinery. Antibodies—Polyclonal antiserum against SNX9 was generated by immunizing a rabbit with glutathione S-transferase (GST)-SNX9 fusion protein. Antibodies were affinity-purified using the recombinant protein or used as serum after removal of anti-GST antibodies. Purchased antibodies were: mouse anti-EEA1 clone 14 (BD Transduction Laboratories, Lexington, KY), mouse anti-β1/β2-adaptin clone 100/1, mouse anti-FLAG (Sigma), rabbit anti-dynamin-2 (Affinity Bioreagents, Golden, CO), mouse anti-dynamin Hudy 1 (Upstate Biotechnology, Waltham, MA), fluorescein-linked donkey anti-rabbit immunoglobulins, and Cy3-linked goat anti-mouse immunoglobulins (Amersham Biosciences). All other antibodies were as described previously (9Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar). DNA Constructs—cDNAs encoding human full-length SNX9, SH3, LC, LC-2, LC-3, and LC-4 were amplified by PCR using the appropriate primers and IMAGE clone 3832234 (UK Human Genome Mapping Project Resource Centre) as template. After digestion, the PCR products were the or (Amersham fusion to and were from full-length SNX9 or from the and the or and were by using the and were described (9Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar). The encoding human dynamin-2 J. R. H. P. 2000; PubMed Scopus Google was a from in K562 cells, DNA encoding SNX9 and was from IMAGE clone 3832234 and the proteins, to a were the DNA encoding SNX9, and were the in HeLa cells, and was from the and the All and were by All were from DNA for of K562 cells by were as described previously U. H. A. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of by in a SNX9 as by HeLa cells in with 2 and were using the C.A. H. PubMed Scopus Google Scholar). and cells on were in in for on and and in serum in were with the appropriate and antibodies and with a laser with an of were for the and to of the of cells were and the number of and was in In 14 and from cells were for and the was was by a of in and uptake cells were in for at and for at with with were in to After cells were and as with and antibodies and (Amersham Biosciences). After cells were and using a of uptake by was as described Schmid S.L. PubMed Scopus Google using glutathione for of and fusion proteins were in and as described previously (9Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar). was and to Ref. J. R. H. P. 2000; PubMed Scopus Google was at with (Amersham to the followed by with of clathrin cytosol was with to was previously using After of the in in and 1 bound was with in the was to using and of cells were and in and in with both with were with of containing 1 and a (9Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar), and the was in After the cells were by at for The of the cells was not by this that structures were The of both SNX9 and Dyn2 was the The was at for and the cytosol was and at The protein was The cytosol was and for at cytosol was to protein (Amersham to antibodies were previously and the were by for at were in in by with or with fusion proteins were as described (9Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar). with proteins, of fusion proteins were used together with 3 AP-2 or clathrin cytosol was on of a in 1 1 and at in a at for In a of serum and as was were from the A was by in of from the cytosol containing and vesicles was in in and for on and at for The was and and the was in two that were in a low and 1 or in an of 1 and After on for the were at for and the were The of the protein in the and were found to be of and SNX9. after of K562 cells as were to by in a in After in an at for were from the and by and and for by A was by in 1 of by from were and proteins were with 1 of and on a as described M. H. Biochem. PubMed Scopus Google Scholar). was for both dynamin-2 and clathrin were from or from of were in as described W. A. Biochemistry. 2001; PubMed Scopus Google and with fusion proteins in a of of containing serum After on for the were by at for were with of and for of with SNX9 previously described that SNX9 contains sites for the appendages of AP-2 (9Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google Scholar). SNX9 of domains with it was of to identify other interacting endocytic proteins to a role for SNX9 in this and to the interaction sites in SNX9. SNX9 and were as fusion proteins and used in and for the of antibodies. 1 the and the used in the present In a from cytosol, proteins were that specifically interacted with the fusion proteins of the major proteins, at and were identified by as clathrin and 1 and of these proteins were by the of SNX9 of proteins interacting with domains of SNX9 and of of proteins at the of or was bound to and with cytosol or After bound proteins were by and by or by using antibodies against clathrin and AP-2 as of proteins to of SNX9. were with or cytosol, and the was with 1 and 2 were identified by as clathrin and the fusion proteins are degradation of clathrin and Dyn2 from cytosol and recombinant domains from Dyn2 and appendage domain of AP-2 to of SNX9. of appendage domain of AP-2 to full-length SNX9 and of the region of SNX9. of clathrin from cytosol to full-length SNX9 and of the region of SNX9 and to the carboxyl-terminal part of as a A of clathrin by is shown to the clathrin clathrin identify the domains in SNX9 that bound and were with fusion proteins containing of SNX9 together with both cytosol and that the SH3 domain in SNX9 binds directly to the in was other proteins interact with dynamin through this region S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (356) Google Scholar). clathrin and AP-2 bind to a region located the SH3 and PX The of the was to the to full-length SNX9, and clathrin-binding was only with the also that the sites for clathrin and AP-2 is in full-length SNX9. The low complexity the SH3 and PX domains that is located the region at amino not to sites for clathrin and AP-2 this was Mapping of for AP-2 and in SNX9 of the specificity of and clathrin to the region is shown in and D. The appendages of AP-2 are known to bind and sequences in proteins D.J. Curr. Opin. Cell Biol. 2000; PubMed Scopus Google Scholar). SNX9 contains a at amino we this to be involved in the of As be in some of to and containing the was a was revealed the fusion protein was to amino The region of amino containing the has of a described for 2002; 10: Full Text Full Text PDF PubMed Scopus Google Scholar). contains the but not the the as and the two described are of was shown by the for the in of in the were to in and a cooperative of the to the is a of the not show of the The region the two may be of for the of the in the fusion protein or may a clathrin were used to a interaction with the region in SNX9 and for of the specificity the of an cooperative was the full-length of showed and interaction. The of the that is by clathrin is not SNX9 not a as present in the of in many of clathrin sequences have been Cell Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). sites in SNX9 and are to one of the clathrin-binding sequences in V.I. De Camilli P. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and low interactions of the with these sites may in cooperative It that clathrin and AP-2 sites in SNX9, may have for the in of a in of SNX9 for Dyn2 an the two proteins in a complex in was shown by from cytosol with antibodies against both Dyn2 and SNX9 As a of is accessory protein involved in endocytosis (14Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (426) Google Scholar), was not in the of Dyn2 are in complex with SNX9 in cytosol was by the that Dyn2 was as a major after with and of the with In addition, an unidentified protein at was specifically by As from the of the it that the of the complex are present in a of cytosol showed that of SNX9 and part of Dyn2 with a of 14.5 S. a complex this to a of of after revealed that also the 41-kDa protein with SNX9 and Dyn2 not the are consistent with the presence of a complex of two molecules of SNX9 Dyn2 and SNX9 on previous study (9Lundmark R. Carlsson S.R. Biochem. J. 2002; 362: 597-607Crossref PubMed Scopus (50) Google we found by cell that of SNX9 was present in the cytosol with on the We have found that the membrane of SNX9 is on the in the cell is at a as in the present study, at of SNX9 an the from the cells after a was by and All molecules SNX9, were in the membrane of the with a large part of the proteins but not of the that AP-2 for was with an that in and SNX9 are molecules were by the in the or by using a of of in showed that clathrin and AP-2 at a of whereas Dyn2 was present on at SNX9 a in the the of both and at part of It may be that SNX9 is at the membrane, with with or with It is that the both from the plasma membrane possibly also together with The of clathrin and AP-2 in both shown in is consistent with their presence on clathrin-coated and of SNX9 to SNX9 has on its we the of recombinant full-length SNX9 and of SNX9 to for of to of we found SNX9 to be in its specificity A and SNX9 bound to phosphoinositides in the and in was It is that SNX9 at one group for was The region for the interaction with is the PX domain. of SNX9 were for only PX were for The in also that of fusion proteins is not to and and together with the in by we have not been to the PX domain in an We therefore not the that the domain the or of the PX domain. of SNX9 in HeLa SNX9 both with and with proteins that the of the endocytic it was of to be with these were with HeLa cells that are to K562 cells SNX9 in a or close the of the cell to the the plasma membrane, see and In this was revealed with both AP-2 and by showed that and of the co-localized with AP-2 and a of the structures were for SNX9 and the presence of of at the plasma membrane. At we out the of structures that are for both AP-2 and SNX9 showed of with at the with EEA1 at early or with at and not that for the of and the was for of the and in these were of by Overexpression of SNX9 of SNX9 to with endocytic proteins and in and in for a role of this protein in endocytosis. this in cell we full-length and of SNX9 in K562 cells and HeLa cells and for the on uptake of full-length SNX9 from the terminus and the uptake of In of SNX9 with a from the amino terminus in or The from was by of uptake in K562 cells with two of the by a cells showed a uptake of and a after of The results for for 2 showed that the of endocytosis is as found by In of truncated containing the sites for and but not full-length SNX9 containing the interfered with the endocytic the results SNX9 as a molecule that participates in the endocytosis in The present study SNX9 as a molecule that in from other sorting nexin family SNX9 to have its role at the plasma membrane and not on and it with the of the endocytic and and with both AP-2 and In addition, its PX domain to specificity of other consistent with their role at the endosomal membrane, have a for C.A. Dixon J.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 919-931Crossref PubMed Scopus (335) Google Scholar, M. Emr S.D. 2001; PubMed Scopus Google Scholar). important in this study was the presence in the cytosol of a complex SNX9 and a complex that also a protein of It that Dyn2 is in SNX9 in the cell in a of Dyn2 to the of the as whereas the of SNX9 with Dyn2 in the we have found that the complex is and the cytosol is in of the in cytosol the complex to some in the and but the relative of SNX9 and Dyn2 that are in the complex are to for this we that at of Dyn2 is with SNX9. and S. R. Work is to identify the and to the role of the It is that SNX9 a and a to and membrane and we at a large of SNX9 at the plasma membrane or on vesicles derived from Dyn2 may be recruited together with SNX9 to the plasma membrane through interactions the PX domain and membrane and the two molecules with clathrin and SNX9 may together with AP-2 and clathrin after formation of the whereas Dyn2 is (15Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (584) Google Scholar, 16Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (356) Google Scholar). In with this a study showed a of SNX9 in a of clathrin-coated vesicles J. A. K. Mol. Biol. Cell. 2003; PubMed Scopus Google Scholar). The that the of clathrin and the of AP-2 to the low complexity region of SNX9 is in the full-length molecule that the of this region in SNX9 may be SNX9 in the cytosol or AP-2 or clathrin and not The interaction with these molecules may be to only at sites of the interaction of SNX9 with the membrane may be after of the SNX9 to of its in the The role of SNX9 in the endocytic process be at present and The of truncated forms of SNX9 on the uptake of may have As SNX9 has for and of have critical in endocytosis, of of these molecules from the cytosol is to have a on the uptake of full-length SNX9, to have the to bind Dyn2 as the truncated not endocytosis. together with the that Dyn2 to be in SNX9, against the of the other full-length SNX9 has its sites for clathrin and AP-2 it is that with sites may bind to both clathrin and AP-2 in the only at the membrane, to or of clathrin and It is also that for SNX9 to be in endocytosis, it the carboxyl-terminal domain. in that this domain is only found in SNX9 and and the function is The amino show a for and it is that this part is essential for the assembly of a complex of SNX9. SNX9 is present in a complex with Dyn2 in the cytosol, SNX9 may play at the later stages of vesicle formation together with It is that in dynamin is suggested to be involved in processes not directly with its established role for endocytic M.A. H. Y. Biochem. Sci. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that dynamin participates in the of both at the sites of endocytosis and De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, Rev. 2002; PubMed Scopus Google Scholar). It is that together with SH3 proteins, is part of a for formation of by the Several proteins are suggested to dynamin to distinct sites of and SNX9 may be involved in this we show that of dynamin in the cytosol are in a complex with SNX9. have shown that SNX9 through its SH3 domain bind to syndrome protein (6Worby C.A. Simonson-Leff N. Clemens J.C. Kruger R.P. Muda M. Dixon J.E. J. Biol. Chem. 2001; 276: 41782-41789Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and S. R. is a major of It is that SNX9 in is involved in or in the endocytic process in the present We and for with the for with for and for