Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing

Abstract

Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2 synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands. Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2 synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands. The bioactive sphingolipid ceramide is involved in the regulation of a wide variety of cellular processes, including apoptosis, autophagy, and cell cycle progression in cancer (1.Galadari S. Rahman A. Pallichankandy S. Thayyullathil F. Tumor suppressive functions of ceramide: evidence and mechanisms.Apoptosis. 2015; 20: 689-711Crossref PubMed Scopus (69) Google Scholar, 2.Morad S.A. Cabot M.C. Ceramide-orchestrated signalling in cancer cells.Nat. Rev. Cancer. 2013; 13: 51-65Crossref PubMed Scopus (428) Google Scholar, 3.Saddoughi S.A. Ogretmen B. Diverse functions of ceramide in cancer cell death and proliferation.Adv. Cancer Res. 2013; 117: 37-58Crossref PubMed Scopus (125) Google Scholar). Ceramide has also been implicated in a number of disease states, including inflammation and inflammatory disorders (4.Maceyka M. Spiegel S. Sphingolipid metabolites in inflammatory disease.Nature. 2014; 510: 58-67Crossref PubMed Scopus (742) Google Scholar) and neurodegenerative diseases (5.Mencarelli C. Martinez-Martinez P. Ceramide function in the brain: when a slight tilt is enough.Cell. Mol. Life Sci. 2013; 70: 181-203Crossref PubMed Scopus (153) Google Scholar). Despite the wide range of processes regulated by ceramide, the precise molecular mechanisms by which ceramide acts as a signaling molecule are not clear. It has been suggested that plasma membrane ceramide acts to stabilize lipid rafts, which act as platforms for the concentration of signaling molecules (6.Grassme H. Jekle A. Riehle A. Schwarz H. Berger J. Sandhoff K. Kolesnick R. Gulbins E. CD95 signaling via ceramide-rich membrane rafts.J. Biol. Chem. 2001; 276: 20589-20596Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar, 7.Grassmé H. Jendrossek V. Bock J. Riehle A. Gulbins E. Ceramide-rich membrane rafts mediate CD40 clustering.J. Immunol. 2002; 168: 298-307Crossref PubMed Scopus (219) Google Scholar). Another possible mechanism of ceramide signaling is through direct interaction with proteins. relatively few direct protein with ceramide been of proteins that are regulated by direct ceramide binding B. S. S. S. M. H. Kolesnick R. of is protein Full Text Full Text PDF PubMed Scopus Google A. J. R. M. S. H. J. and protein as a protein Sci. PubMed Scopus Google protein J. M. Ceramide directly protein to a protein signaling Biol. Chem. Full Text Full Text PDF PubMed Scopus Google A. S.A. P. S. Ogretmen B. interaction the and ceramide via binding is involved in the regulation of protein and J. PubMed Scopus Google and M. M. C. J. R. P. C. J. M. S. by J. PubMed Scopus Google Scholar). Thus, the of additional ceramide-binding proteins to a of ceramide functions as a signaling Although been used in to for S. B. of molecule yeast cDNA Mol. Biol. PubMed Scopus Google Scholar, S. B. yeast surface human proteome display to identify Mol. Biol. 2015; PubMed Scopus Google Scholar, S. B. of protein yeast surface human proteome Mol. Biol. 2015; PubMed Scopus Google Scholar). The used yeast is when the be the yeast cell and and display is to of proteins. B. J. of direct protein of Chem. Biol. PubMed Scopus Google Scholar, S. V. C. H. for bioactive the in the Chem. 2013; PubMed Scopus Google Scholar) and protein R. M. and protein PubMed Scopus Google Scholar, J. H. of protein Sci. 2013; Scholar) been but also including the in low proteins and of and S. B. yeast surface human proteome display to identify Mol. Biol. 2015; PubMed Scopus Google Scholar). We the and of yeast surface cDNA display to novel protein-ligand S. B. of molecule yeast cDNA Mol. Biol. PubMed Scopus Google Scholar, S. B. of protein yeast surface human proteome Mol. Biol. 2015; PubMed Scopus Google Scholar, S. B. and of a yeast human cDNA library to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. yeast human proteome to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. of yeast cDNA Mol. Biol. PubMed Scopus Google Scholar, S. B. of novel protein-ligand by analysis of yeast surface cDNA library Mol. Biol. 2015; PubMed Scopus Google Scholar, S. B. analysis of yeast surface cDNA library by to identify novel protein-ligand Full Text Full Text PDF PubMed Scopus Google Scholar). we for of human ceramide-binding proteins. deep sequencing to we identified a number of ceramide-binding many of which novel we identified and validated EF-hand and proteins as binding that ceramide may cellular by directly with proteins. The of yeast human cDNA and and for been S. B. of molecule yeast cDNA Mol. Biol. PubMed Scopus Google Scholar, S. B. and of a yeast human cDNA library to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. yeast human proteome to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. of yeast cDNA Mol. Biol. PubMed Scopus Google Scholar, S. B. of novel protein-ligand by analysis of yeast surface cDNA library Mol. Biol. 2015; PubMed Scopus Google Scholar, S. B. analysis of yeast surface cDNA library by to identify novel protein-ligand Full Text Full Text PDF PubMed Scopus Google Scholar, surface display for PubMed Scopus Google Scholar). the yeast display cDNA library of yeast and with at of additional library by the of to a concentration of and the yeast were with at were on A. S. A. and yeast surface Mol. Biol. 2015; PubMed Scopus Google Scholar) and the other on S. B. of molecule yeast cDNA Mol. Biol. PubMed Scopus Google Scholar, S. B. and of a yeast human cDNA library to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. yeast human proteome to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. and yeast cell surface display to identify novel cell human Mol. Biol. 2015; PubMed Scopus (17) Google Scholar). the were by of in to of and with for at The were with with the of an from of the cDNA display library were by with in of to the and for with at the were three with the The and yeast were on yeast and for at to to were by the and to an additional of on the protocol and to the that of the yeast in the binding to ceramide when by at a concentration of not the of ceramide-binding yeast the yeast cDNA display library as and of library with and in of at a concentration of and the with at were with in of with used of fragments calcium with for at and with and the yeast were a cell yeast were on and for at to to were by the and to an additional of the protocol but with and the of for to the of ceramide and ceramide ceramide and were by and with and at a and at for yeast from the were with ceramide and in with for at with and by that were were on and for at to to of fragments ceramide neuronal calcium sensor of the starting library and yeast were with and with for at with with and with at in for at with were with and by a were from the cDNA display and the ceramide-binding and yeast a protocol a cell as S. B. of molecule yeast cDNA Mol. Biol. PubMed Scopus Google Scholar). cDNA for the deep the were used as for the and and The were the The the cDNA from the starting yeast cDNA display library and the ceramide-binding and were by cycle at the The deep sequencing were the R. M. A. J. a analysis for Mol. Biol. PubMed Scopus Google Scholar, B. C. R. P. J. A. a for Res. PubMed Scopus Google Scholar, J. A. J. a approach for and in the Biol. PubMed Scopus Google Scholar). the were by A. J. A. of with PubMed Scopus Google by for C. H. R. of in the of and Biol. 2013; PubMed Scopus Google Scholar) to an which by C. A. and by and cell PubMed Scopus Google Scholar) the to in of fragments The were a candidate of ceramide-binding protein frequency and to library for and for were to the a of cDNA by for in the of candidate the were from the by the to the The were by the to a of The were and used as a for the of candidate cDNA candidate were not the the with the and cDNA for candidate were The the candidate ceramide-binding protein fragments were the yeast display S. B. and of a yeast human cDNA library to identify Full Text Full Text PDF PubMed Scopus Google Scholar) by from the of candidate cDNA and the ceramide-binding as a The were the yeast by a heat shock and were verified by as S. B. and yeast cell surface display to identify novel cell human Mol. Biol. 2015; PubMed Scopus (17) Google Scholar). were in yeast to surface display of the candidate ceramide-binding protein lipids were to yeast were with and with for at with ceramide, and in to surface display The yeast were with and with of and for with the yeast were by a additional lipid binding yeast were with and with for at with ceramide, and The yeast were with and with of and for with the yeast were by a the and and the and were and as HPCA and HPCA were for ceramide binding by as of HPCA and HPCA a method and were used with HPCA as for were with an additional to the were and with via and were on The HPCA and HPCA yeast display were as and of the were with and in of with and and for with at were with in of with and with for at with and yeast were a cell were as and to a of yeast from the were for binding to ceramide by as with ceramide binding were by as S. B. and yeast cell surface display to identify novel cell human Mol. Biol. 2015; PubMed Scopus (17) Google Scholar). mutagenesis used to and and the The of yeast and HPCA for by yeast with of ceramide at with for were with and with at with for with and by a were and were The for HPCA and HPCA were via the mammalian with a were with the HPCA and HPCA mammalian with to the were by and on a were by at and were as and with the were by the with of the HPCA and HPCA cell for at with The were three with the in and by to membrane a by and by with to the were a We the and of yeast surface display human protein fragments from cDNA from human S. B. of molecule yeast cDNA Mol. Biol. PubMed Scopus Google Scholar, S. B. yeast surface human proteome display to identify Mol. Biol. 2015; PubMed Scopus Google Scholar, S. B. of protein yeast surface human proteome Mol. Biol. 2015; PubMed Scopus Google Scholar, S. B. and of a yeast human cDNA library to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. yeast human proteome to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. of yeast cDNA Mol. Biol. PubMed Scopus Google Scholar, S. B. of novel protein-ligand by analysis of yeast surface cDNA library Mol. Biol. 2015; PubMed Scopus Google Scholar, S. B. analysis of yeast surface cDNA library by to identify novel protein-ligand Full Text Full Text PDF PubMed Scopus Google Scholar). identify human proteins with for ceramide, we used these by to create for that display ceramide-binding protein fragments in the we ceramide and used the to and yeast protein fragments with for ceramide. of were and the binding of the starting library and the from the and were for binding to ceramide by The from the of binding to ceramide but binding to the we to ceramide-binding for The of with ceramide. the concentration of ceramide to in the of the and by and binding to ceramide in the for that bind to ceramide, we an additional of with ceramide and and and the The of the by ceramide and and binding to ceramide although binding identify ceramide-binding protein fragments from the we the cDNA by and to deep sequencing estimate of the starting library and a for in the we deep sequencing on the starting library. The and starting library and were and the R. M. A. J. a analysis for Mol. Biol. PubMed Scopus Google Scholar, B. C. R. P. J. A. a for Res. PubMed Scopus Google Scholar, J. A. J. a approach for and in the Biol. PubMed Scopus Google Scholar) to at the of in the human used for a in the for the starting cDNA library. The and ceramide-binding and with that the the three with that a of of by human are in our yeast display human cDNA library. of the starting and is in a candidate of ceramide-binding protein we frequency and to to the these for the and for the We for the cDNA of the from from the and and to protein that were to a protein the and we identified and protein fragment protein fragment that the protein of the of for the 234 is in the 234 were identified in the and and ceramide-binding protein fragments that and in the and sequencing in a we a for domains in the candidate ceramide-binding protein of protein domains were identified in the 234 candidate protein Several of the domains are present in of the 234 candidate ceramide-binding protein fragments and of the that are to EF-hand domains that are the candidate ceramide-binding protein fragments are in Based on the data, we of the candidate ceramide-binding protein fragments for We yeast the candidate ceramide-binding protein fragments and for binding to ceramide, and by and of the protein fragments a of binding to ceramide, and (17) of the protein including of the EF-hand and bound to ceramide the fragments from and binding to lipids other ceramide. Interestingly, the protein fragment bound to ceramide of the other lipids and The protein fragments of binding to ceramide, with the and protein fragments and the interaction and The protein domains in the verified ceramide-binding protein fragments are in and for the and for the protein domains present in the verified ceramide-binding protein HPCAL1, NCS1, HPCAL4, calcium-binding shock chaperonin-binding of lipids and of and type region lipid type region lipid surface for mediate binding to of in a lipid binding we additional which has a and which has a the of lipids used in study is in to ceramide binding to for binding to in with ceramide binding for with the of and of the verified ceramide-binding (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding (NCALD, HPCAL4, and KCNIP3) protein fragments belong to the neuronal calcium sensor family of proteins. proteins in and and are of a of four EF-hand domains S. and of neuronal calcium sensor PubMed Scopus Google Scholar, the of the neuronal calcium sensor PubMed Scopus Google Scholar, and in neuronal calcium 2015; PubMed Scopus Google Scholar, S. of neuronal calcium sensor proteins and for of by Mol. 2014; PubMed Scopus Google Scholar). We the protein with the binding to ceramide, as a for the of important for interaction with ceramide. We a library of yeast HPCA and to HPCA that the to bind ceramide by not of the molecular of the protein by S. of Biol. PubMed Scopus Google which is to human suggested that by the and and the and be capable of binding ceramide We that may the in HPCA to bind ceramide be we yeast the HPCA domains of the and and the and and for binding to ceramide. HPCA and HPCA bound to ceramide We of yeast of HPCA and HPCA and to that the to bind ceramide by we were in at in HPCA and HPCA were to binding to ceramide and We to to a HPCA mutant of ceramide Based on and family and we on at and in HPCA and HPCA which are that were to ceramide binding the of these is to ceramide binding in the of we a yeast HPCA with and and for ceramide with HPCA HPCA bound to ceramide with an of HPCA binding to ceramide The and for ceramide binding are in the family of S. of Biol. PubMed Scopus Google Scholar) and human J. V. P. and of human Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) that and are as the of mammalian cell-produced HPCA to bind to ceramide, we from HPCA and HPCA and used for with ceramide, and HPCA by the but not the the HPCA mutant not by of the Thus, HPCA in mammalian is capable of binding to ceramide, and that we identified and are for Despite the of ceramide as a signaling molecule, relatively few to identify ceramide-binding proteins. ceramide by analysis to identify ceramide-binding proteins A. S.A. P. S. Ogretmen B. interaction the and ceramide via binding is involved in the regulation of protein and J. PubMed Scopus Google Scholar, R. Sandhoff K. of ceramide binding proteins in neuronal a of Res. 2002; PubMed Scopus Google Scholar, V. H. of proteins in PubMed Scopus Google Scholar). of the identified a number of ceramide-binding protein V. H. of proteins in PubMed Scopus Google and of the were verified directly by ceramide binding Although the study by A. S.A. P. S. Ogretmen B. interaction the and ceramide via binding is involved in the regulation of protein and J. PubMed Scopus Google Scholar) identified ceramide-binding of protein ceramide demonstrated to directly bind and interaction with of Several additional ceramide-binding proteins been identified by direct including A. J. R. M. S. H. J. and protein as a protein Sci. PubMed Scopus Google and A. J. ceramide binding to protein and in PubMed Scopus Google M. M. C. J. R. P. C. J. M. S. by J. PubMed Scopus Google A. C. J. binding of ceramide to in and PubMed Scopus Google and of M. H. A. Kolesnick R. ceramide-binding of membrane PubMed Scopus Google Scholar). the number of ceramide-interacting proteins with validated direct binding is with the of and the of ceramide-binding proteins not in the of binding to other ceramide cellular are to identify cellular proteins that bind to ceramide we our yeast surface cDNA library approach to identify protein-ligand interaction on a S. B. and of a yeast human cDNA library to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. yeast human proteome to identify Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. analysis of yeast surface cDNA library by to identify novel protein-ligand Full Text Full Text PDF PubMed Scopus Google Scholar). We used to for ceramide-binding from the yeast surface human proteome display library and the by deep which identified 234 candidate ceramide-binding protein direct of ceramide-binding protein fragments were many of which novel the ceramide-binding protein the protein bound ceramide and not of the other lipids when at a concentration of at a concentration of binding to and for protein although binding to for proteins with the of proteins the interaction with ceramide. The of binding is and suggests that may be to the of these proteins. of the verified ceramide-binding protein fragments bound to of lipids in to ceramide when at a concentration of The protein fragment bound to and although the binding binding to ceramide. The protein fragment also bound to and Interestingly, the protein fragment in that bound to functions of in the prostaglandin and of to is to be by binding P. Ceramide is for the of and prostaglandin Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, is possible that binding by and prostaglandin by the in membrane The domains in these protein fragments and and are to be involved in lipids other molecules lipid binding is not Based on the binding for the can be the of the lipids that The the ceramide and molecules used in study is a binding is to the protein fragments is that the of the with binding to the lipids with and is also binding to ceramide. Although the is and ceramide, binding to of the protein fragments with the of The binding of the ceramide has been to be by a the and of ceramide and in K. S. M. K. R. for lipid by for of Sci. PubMed Scopus Google Scholar). were in with in a interaction K. S. M. K. R. for lipid by for of Sci. PubMed Scopus Google Scholar). Thus, the for ceramide that we for of the ceramide-binding protein fragments may be the of a binding mechanism to that for the used in study has the other lipids in the binding be a our that the of the lipid is an important that binding although that and may also important additional lipid the important for binding can be It be that the lipids used in study are not to cellular ceramide, which in many in the a by a with which may including of our are in at lipid to the It is that the lipids may as at concentration in although has not been lipids as the lipids used in study are used for binding The ceramide used in study has been in H. M. H. K. of ceramide in the of PubMed Scopus Google Scholar). the of protein domains in the 234 candidate ceramide-binding protein fragments including domains in the verified ceramide-binding protein fragments The is the calcium-binding EF-hand domain, which in in of the 234 including of the verified ceramide-binding protein four of the ceramide-binding protein fragments HPCAL1, NCS1, and as as three additional and HPCAL4, belong to the family of proteins. are mammalian proteins S. and of neuronal calcium sensor PubMed Scopus Google Scholar, and in neuronal calcium 2015; PubMed Scopus Google and are in the and are involved in cellular processes, including R. S. K. M.C. regulation of PubMed Scopus Google M. A. J. K. S. calcium and PubMed Scopus Google of and to the and Sci. PubMed Scopus Google Scholar, M. K. and to the of the J. Full Text Full Text PDF PubMed Scopus Google R. neuronal calcium interaction for 13: PubMed Scopus Google regulation functions in an in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. K. E. of by neuronal calcium 2014; PubMed Scopus Google Scholar, of binding of neuronal calcium to the calcium 2014; PubMed Scopus Google and K. J. calcium of calcium for PubMed Scopus Google Scholar, M. C. F. A. K. K. of by neuronal calcium and in PubMed Scopus Google Scholar, J. A. for in function and PubMed Scopus Google and sensor the mammalian of is in and and from Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. E. M. S. S. J. A. calcium binds to regulated and functions in and in Sci. 2002; PubMed Google Scholar, A. K. J. in by neuronal in 2002; PubMed Google Scholar, M. S. J. A. S. is a of neuronal calcium in in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. F. J. A. calcium neuronal through PubMed Scopus Google Scholar, J. C. K. J. S.A. B. A. calcium in through of Sci. PubMed Scopus Google Scholar, J. A. S. A. of neuronal calcium and interaction in regulated of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). proteins are of four EF-hand the of which does not bind of proteins is a that has been to be directly involved in interaction for proteins S. and of neuronal calcium sensor PubMed Scopus Google Scholar, and in neuronal calcium 2015; PubMed Scopus Google Scholar). Interestingly, the HPCA we identified that ceramide binding in that binding of ceramide to proteins interaction with other be to the of the identified of the identified ceramide-binding protein fragments are to mediate to the the STI1, tetratricopeptide repeat and domains are to be involved in protein Thus, is possible that ceramide binding by these domains may act to these protein and Another mechanism of regulation is through protein The of ceramide to with a range of protein be the for in cellular sequencing is an method to library and S. S. F. C. deep sequencing to 2014; PubMed Scopus Google Scholar, A. P. S. C. J. A. J. P. J. J. A. and through library yeast display and Mol. Biol. 2015; PubMed Scopus Google Scholar). The of yeast surface human display with deep sequencing of the is a method for the of protein-ligand We used method to identify 234 candidate ceramide-binding protein of which were the number of is that additional of ceramide-binding proteins. method the of non-protein as lipids and which with yeast The used method for lipid and proteins is of by Although method is the to be by proteins that are in the are and to but the and to protein has yeast cDNA with deep sequencing the of low we identified ceramide-binding protein fragments that were present at low in the starting library starting library and starting library It is that these be identified sequencing for the yeast cDNA display library and for of the in the human used for of the human It is that the number of by the deep sequencing not to the of the library. is by the that many with an of in the library were to in the Thus, the library is by the deep sequencing library the of the yeast display the of important for binding interactions, as demonstrated by our of HPCA be at the of the identified the of the HPCA mutant at the of ceramide binding in HPCA the family proteins are particularly the identified that are for ceramide we that of the in other ceramide-binding proteins ceramide binding as possible to with additional family proteins. the of the yeast surface cDNA display/deep sequencing the approach to other to a map of We R. for with lipid We R. and for with