The Membrane Domains Occupied by Glycosylphosphatidylinositol-anchored Prion Protein and Thy-1 Differ in Lipid Composition

Abstract

Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or “rafts” on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (≤3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained ∼20% of prion protein. of prion protein and Thy-1 DRMs was In the was with Thy-1 and prion protein DRMs and the were protein DRMs contained Thy-1 DRMs and had levels of in keeping with the and of the two proteins and show the DRMs and of of of Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or “rafts” on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (≤3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained ∼20% of prion protein. of prion protein and Thy-1 DRMs was In the was with Thy-1 and prion protein DRMs and the were protein DRMs contained Thy-1 DRMs and had levels of in keeping with the and of the two proteins and show the DRMs and of of of of a In to with detergent-resistant prion detergent-resistant prion and to of in a “rafts” the microdomains the and of of the domains in non-ionic to detergent-resistant membranes (DRMs) that proteins both of the proteins the and the proteins the and of prion protein of a for the of to the associated with prion to on the neuronal in domains to Thy-1, the protein of differences in in the and of Thy-1 the of to the of and a of a of a few and on their in non-ionic to domains on the surface, and and of Thy-1 a that differences in in the the two of this to the in and Thy-1 to the of proteins in that found on the of the the the and a of of the detergent the that their separate and and that the detergent for of domains a found with the non-ionic Brij 96 Brij 96 to a in both and Thy-1 on neuronal of DRMs a of were in the were for with the in was in with in with was of was in 1% Brij 96 in for a was in and with of a in Brij 96 and in a were the of the was for and Thy-1, and of the with the of both proteins was to immunoaffinity using to the were and and for Thy-1, were the was two or Thy-1 was was two with and of a with of Thy-1 the was with and of to a with of was for and the was and to were using the was and were on a and with in the of were to the of and were in and for was on a with a was a of was to of and was to of and was for a of was for a of and was for a of in was a of and a of for a of was with in for of a of and a of were of and were for and were and and were were was to of was of with that were in for were for with or of to the was in a to to the was to a of were in 1% in and for in a were to the of clustered for both and that was of label on the was a using for Thy-1 or showed label for the protein. the of for Thy-1 that of label were a of of that for label on of and Thy-1 on the and in the to for and Thy-1 separate microdomains on the neuronal surface, were with to or of and in the for protein was in clusters largely of the or two immunogold for were found the of Thy-1 clusters and and Thy-1 label was found a of the of and Thy-1, a was were of a the a of for the to the or two molecules of Thy-1 of both and Thy-1 of immunogold of and of Thy-1 in clusters containing immunogold in was found clusters of immunogold that contained no the of label associated with Thy-1 clusters of the a few of label were associated with Thy-1 that 40% of the contained containing label for both proteins in in the few clusters or contained immunogold for both proteins of of and Thy-1 label on a of label for and Thy-1 clusters in the clusters of immunogold and clusters in the clusters of Thy-1 immunogold and clusters containing label for both proteins in the immunogold for Thy-1 and the a with for clusters of to the of and Thy-1 on the neuronal in that found with to a of whereas a of Thy-1 DRMs associated with In to for a few molecules of the to a in keeping with the of of and Thy-1 of DRMs with of with Thy-1, whereas of the Thy-1 with of this to the of DRMs in or Thy-1 was the was in and was with the or Thy-1 were with of to the of DRMs the levels of Thy-1, and was with DRMs that contained 40% of the with of the Thy-1, to and of Thy-1 DRMs were that contained of the Thy-1 the of the associated with of in the Thy-1 DRMs a with associated with the of and Thy-1 the of and Thy-1 a in Brij the and the 40% was the that contained the levels of and in and Thy-1 in the and the Thy-1 or to the for and Thy-1 protein in the in and Thy-1 of the of and Thy-1 and the of DRMs were the levels of the was a for the and the of of and to the were were and the was the for that of the in Thy-1, and DRMs in a of the the of that of were the that were with the differences in for the of the two DRMs and of and the were of DRMs was that of Thy-1 although both were the of the was with although the of this in Thy-1 DRMs was in the whereas DRMs were the levels of and in DRMs were that Thy-1 DRMs Thy-1 DRMs had levels of or and both Thy-1 and had levels of the In was in Thy-1 DRMs and and of in and to the in a and DRMs in the to and to for the in of their in the in of the Thy-1 DRMs were in the to and and DRMs had a of to and for and the differences to a in the of for for Thy-1 and DRMs in the the differences in the of of and the of in the two of a of the of and to the the for the were for the of in the and in the Thy-1 DRMs were with in DRMs had and Thy-1 DRMs this of Thy-1 neuronal of the to and in and Thy-1 in a of in the Thy-1 and of or that or or of for all of in in the differences for Thy-1 and DRMs using the in the for of in Thy-1 and in a Thy-1 and was in the both and in was in Thy-1 DRMs the were on was in DRMs of both Thy-1 of all and of all In the had a of in Thy-1, and in of the of was in DRMs with a of this of the associated with were with in Thy-1 was no in the to in Thy-1 and DRMs was a of the in of was in of the of the to the and of or with the in Thy-1 or of the differences proteins the in their a to the of neuronal for the and clustered in microdomains on the proteins in their detergent with Thy-1 and on that differences in the of proteins and Thy-1 on the a of the of of and Thy-1 on the of the separate of proteins using immunogold to that had or with for a immunogold was to and largely of both proteins was of had no Thy-1 label 40% of Thy-1 in clusters that had a few associated molecules of although 1% of clusters were with immunogold for both of of the of and Brij 96 Thy-1 and the with of immunoaffinity for of the of the of DRMs containing Thy-1 with a few molecules of were In the of detergent of membranes to this clusters of both and Thy-1 show in and to on the neuronal DRMs that the of Thy-1 and a of was to the of DRMs containing levels of the protein in the Thy-1 or containing the levels of Thy-1 the were and for Thy-1 to the of DRMs in or proteins were of DRMs that the and the of of or Thy-1 in the DRMs of the proteins that the appreciable in Thy-1 of and Thy-1 DRMs were the was on the of of DRMs was the of the that Brij 96 the neuronal that the of and separate all with the for of Thy-1 and was appreciable of the protein in differences and Thy-1 DRMs to of the in Thy-1 DRMs the in the immunoaffinity a the of the of Thy-1 and DRMs the to protein with to that the of the of that to the of the of in the of of the Thy-1 levels of found using a of in the that and levels of in DRMs and of for in with the in both the and in of the of with the to with the the very little with was a of in the in in were of the levels of of were found in DRMs of the a that the was and of in domains the of this protein to and of for that and in largely in the and their to membranes the of of of that showed in the of of and and DRMs the of a had the the in the of Thy-1 and DRMs was of of in this for Thy-1 and and and and and in the this was to and in DRMs with in Thy-1 was the levels in the differences in to the of proteins Thy-1 and of this and in Thy-1 in the and Thy-1 DRMs showed a of very to were the to two of although the in separate or in to a the this the of the a In the of of the to to the and of the and with to with the to in the that very levels of that to the a the of the of the the of the DRMs their the of and their with proteins that the that that to the on to that to the to the of neuronal in to the of in and Thy-1 of their with to of the of the of to the of and of a In to with detergent-resistant prion detergent-resistant prion and to of in a “rafts” the microdomains the and of of the domains in non-ionic to detergent-resistant membranes (DRMs) that proteins both of the proteins the and the proteins the and of prion protein of a for the of to the associated with prion to on the neuronal in domains to Thy-1, the protein of differences in in the and of Thy-1 the of to the of and a of a of a few and on their in non-ionic to domains on the surface, and and of Thy-1 a that differences in in the the two of this to the in and Thy-1 to the of proteins in that found on the of the the the and a of of the detergent the that their separate and and that the detergent for of domains a found with the non-ionic Brij 96 Brij 96 to a in both and Thy-1 on neuronal of DRMs a of were in the were for with the in was in with in with was of was in 1% Brij 96 in for a was in and with of a in Brij 96 and in a were the of the was for and Thy-1, and of the with the of both proteins was to immunoaffinity using to the were and and for Thy-1, were the was two or Thy-1 was was two with and of a with of Thy-1 the was with and of to a with of was for and the was and to were using the was and were on a and with in the of were to the of and were in and for was on a with a was a of was to of and was to of and was for a of was for a of and was for a of in was a of and a of for a of was with in for of a of and a of were of and were for and were and and were were was to of was of with that were in for were for with or of to the was in a to to the was to a of were in 1% in and for in a were to the of clustered for both and that was of label on the was a using for Thy-1 or showed label for the protein. the of for Thy-1 that of label were a of of that for label on of DRMs a of were in the were for with the in was in with in with was of was in 1% Brij 96 in for a was in and with of a in Brij 96 and in a were the of the was for and Thy-1, and of the with the of both proteins was to immunoaffinity using to the were and and for Thy-1, were the was two or Thy-1 was was two with and of a with of Thy-1 the was with and of to a with of was for and the was and to were using the was and were on a and with in the of were to the of and were in and for was on a with a was a of was to of and was to of and was for a of was for a of and was for a of in was a of and a of for a of was with in for of a of and a of were of and were for and were and and were were was to of was of with that were in for were for with or of to the was in a to to the was to a of were in 1% in and for in a were to the of clustered for both and that was of label on the was a using for Thy-1 or showed label for the protein. the of for Thy-1 that of label were a of of that for label on of and Thy-1 on the and in the to for and Thy-1 separate microdomains on the neuronal surface, were with to or of and in the for protein was in clusters largely of the or two immunogold for were found the of Thy-1 clusters and and Thy-1 label was found a of the of and Thy-1, a was were of a the a of for the to the or two molecules of Thy-1 of both and Thy-1 of immunogold of and of Thy-1 in clusters containing immunogold in was found clusters of immunogold that contained no the of label associated with Thy-1 clusters of the a few of label were associated with Thy-1 that 40% of the contained containing label for both proteins in in the few clusters or contained immunogold for both proteins the of and Thy-1 on the neuronal in that found with to a of whereas a of Thy-1 DRMs associated with In to for a few molecules of the to a in keeping with the of of and Thy-1 of DRMs with of with Thy-1, whereas of the Thy-1 with of this to the of DRMs in or Thy-1 was the was in and was with the or Thy-1 were with of to the of DRMs the levels of Thy-1, and was with DRMs that contained 40% of the with of the Thy-1, to and of Thy-1 DRMs were that contained of the Thy-1 the of the associated with of in the Thy-1 DRMs a with associated with the of and Thy-1 the of and Thy-1 a in Brij the and the 40% was the that contained the levels of and in and Thy-1 in the and the Thy-1 or to the for and Thy-1 protein in the in and Thy-1 of the of and Thy-1 and the of DRMs were the levels of the was a for the and the of of and to the were were and the was the for that of the in Thy-1, and DRMs in a of the the of that of were the that were with the differences in for the of the two DRMs and of and the were of DRMs was that of Thy-1 although both were the of the was with although the of this in Thy-1 DRMs was in the whereas DRMs were the levels of and in DRMs were that Thy-1 DRMs Thy-1 DRMs had levels of or and both Thy-1 and had levels of the In was in Thy-1 DRMs and and of in and to the in a and DRMs in the to and to for the in of their in the in of the Thy-1 DRMs were in the to and and DRMs had a of to and for and the differences to a in the of for for Thy-1 and DRMs in the the differences in the of of and the of in the two of a of the of and to the the for the were for the of in the and in the Thy-1 DRMs were with in DRMs had and Thy-1 DRMs this of Thy-1 neuronal of the to and in and Thy-1 in a of in the Thy-1 and of or that or or of for all of in in the differences for Thy-1 and DRMs using the in the for of in Thy-1 and in a Thy-1 and was in the both and in was in Thy-1 DRMs the were on was in DRMs of both Thy-1 of all and of all In the had a of in Thy-1, and in of the of was in DRMs with a of this of the associated with were with in Thy-1 was no in the to in Thy-1 and DRMs was a of the in of was in of the of the to the and of or with the in Thy-1 or of the differences of and Thy-1 on the and in the to for and Thy-1 separate microdomains on the neuronal surface, were with to or of and in the for protein was in clusters largely of the or two immunogold for were found the of Thy-1 clusters and and Thy-1 label was found a of the of and Thy-1, a was were of a the a of for the to the or two molecules of Thy-1 of both and Thy-1 of immunogold of and of Thy-1 in clusters containing immunogold in was found clusters of immunogold that contained no the of label associated with Thy-1 clusters of the a few of label were associated with Thy-1 that 40% of the contained containing label for both proteins in in the few clusters or contained immunogold for both proteins the of and Thy-1 on the neuronal in that found with to a of whereas a of Thy-1 DRMs associated with In to for a few molecules of the to a in keeping with the of of and Thy-1 of DRMs with of with Thy-1, whereas of the Thy-1 with of In this to the of DRMs in or Thy-1 was the was in and was with the or Thy-1 were with of to the of DRMs the levels of Thy-1, and was with DRMs that contained 40% of the with of the Thy-1, to and of Thy-1 DRMs were that contained of the Thy-1 the of the associated with of in the Thy-1 DRMs a with associated with the in and Thy-1 of the of and Thy-1 and the of DRMs were the levels of the was a for the and the of of and to the were were and the was the for that of the in Thy-1, and DRMs in a of the the of that of were the that were with the differences in for the of the two DRMs and of and the were of DRMs was that of Thy-1 although both were the of the was with although the of this in Thy-1 DRMs was in the whereas DRMs were the levels of and in DRMs were that Thy-1 DRMs Thy-1 DRMs had levels of or and both Thy-1 and had levels of the In was in Thy-1 DRMs and and to the and DRMs in the to and to for the in of their in the in of the Thy-1 DRMs were in the to and and DRMs had a of to and for and the differences to a in the of for for Thy-1 and DRMs in the the differences in the of of and the of in the two of a of the of and to the the for the were for the of in the and in the Thy-1 DRMs were with in DRMs had and Thy-1 DRMs this of Thy-1 neuronal Thy-1 and was in the both and in was in Thy-1 DRMs the were on was in DRMs of both Thy-1 of all and of all In the had a of in Thy-1, and in of the of was in DRMs with a of this of the associated with were with in Thy-1 was no in the to in Thy-1 and DRMs was a of the in of was in proteins the in their a to the of neuronal for the and clustered in microdomains on the proteins in their detergent with Thy-1 and on that differences in the of proteins and Thy-1 on the a of the of of and Thy-1 on the of the separate of proteins using immunogold to that had or with for a immunogold was to and largely of both proteins was of had no Thy-1 label 40% of Thy-1 in clusters that had a few associated molecules of although 1% of clusters were with immunogold for both of of the of and Brij 96 Thy-1 and the with of immunoaffinity for of the of the of DRMs containing Thy-1 with a few molecules of were In the of detergent of membranes to this clusters of both and Thy-1 show in and to on the neuronal DRMs that the of Thy-1 and a of was to the of DRMs containing levels of the protein in the Thy-1 or containing the levels of Thy-1 the were and for Thy-1 to the of DRMs in or proteins were of DRMs that the and the of of or Thy-1 in the DRMs of the proteins that the appreciable in Thy-1 of and Thy-1 DRMs were the was on the of of DRMs was the of the that Brij 96 the neuronal that the of and separate all with the for of Thy-1 and was appreciable of the protein in differences and Thy-1 DRMs to of the in Thy-1 DRMs the in the immunoaffinity a the of the of Thy-1 and DRMs the to protein with to that the of the of that to the of the of in the of of the Thy-1 levels of found using a of in the that and levels of in DRMs and of for in with the in both the and in of the of with the to with the the very little with was a of in the in in were of the levels of of were found in DRMs of the a that the was and of in domains the of this protein to and of for that and in largely in the and their to membranes the of of of that showed in the of of and and DRMs the of a had the the in the of Thy-1 and DRMs was of of in this for Thy-1 and and and and and in the this was to and in DRMs with in Thy-1 was the levels in the differences in to the of proteins Thy-1 and of this and in Thy-1 in the and Thy-1 DRMs showed a of very to were the to two of although the in separate or in to a the this the of the a In the of of the to to the and of the and with to with the to in the that very levels of that to the a the of the of the the of the DRMs their the of and their with proteins that the that that to the on to that to the to the of neuronal in to the of in and Thy-1 of their with to of the of the of to the of and proteins the in their a to the of neuronal for the and clustered in microdomains on the proteins in their detergent with Thy-1 and on that differences in the of proteins and Thy-1 on the a of the of of and Thy-1 on the of the separate of proteins using immunogold to that had or with for a immunogold was to and largely of both proteins was of had no Thy-1 label 40% of Thy-1 in clusters that had a few associated molecules of although 1% of clusters were with immunogold for both of of the of and Brij 96 Thy-1 and the with of immunoaffinity for of the of the of DRMs containing Thy-1 with a few molecules of were In the of detergent of membranes to this clusters of both and Thy-1 show in and to on the neuronal DRMs that the of Thy-1 and a of was to the of DRMs containing levels of the protein in the Thy-1 or containing the levels of Thy-1 the were and for Thy-1 to the of DRMs in or proteins were of DRMs that the and the of of or Thy-1 in the DRMs of the proteins that the appreciable in Thy-1 of and Thy-1 DRMs were the was on the of of DRMs was the of the that Brij 96 the neuronal that the of and separate all with the for of Thy-1 and was appreciable of the protein in differences and Thy-1 DRMs to of the in Thy-1 DRMs the in the immunoaffinity a the of the of Thy-1 and DRMs the to protein with to that the of the of that to the of the of in the of of the Thy-1 levels of found using a of in the that and levels of in DRMs and of for in with the in both the and in of the of with the to with the the very little with was a of in the in in were of the levels of of were found in DRMs of the a that the was and of in domains the of this protein to and of for that and in largely in the and their to membranes the of of of that showed in the of of and and DRMs the of a had the the in the of Thy-1 and DRMs was of of in this for Thy-1 and and and and and in the this was to and in DRMs with in Thy-1 was the levels in the differences in to the of proteins Thy-1 and of this and in Thy-1 in the and Thy-1 DRMs showed a of very to were the to two of although the in separate or in to a the this the of the a In the of of the to to the and of the and with to with the to in the that very levels of that to the a the of the of the the of the DRMs their the of and their with proteins that the that that to the on to that to the to the of neuronal in to the of in and Thy-1 of their with to of the of the of to the of and and for and for on the with with