Caveolin-1 Regulates the Functional Localization of N-Acetylglucosaminyltransferase III within the Golgi Apparatus

In an investigation of the mechanism underlying the functional sublocalization of glycosyltransferases within the Golgi apparatus, caveolin-1 was identified as a possible cellular factor. Caveolin-1 appears to regulate the localization of N-acetylglucosaminyltransferase III (GnT-III) in the intra-Golgi subcompartment. Structural analyses of total cellular N-glycans indicated that the overexpression of GnT-III in human hepatoma cells, in which caveolin-1 is not expressed, failed to reduce branch formation, whereas expression of caveolin-1 led to a dramatic decrease in the extent of branching with no enhancement in GnT-III activity. Because the addition of a bisecting GlcNAc by GnT-III to the core β-Man in N-glycans prevents the action of GnT-IV and GnT-V, both of which are involved in branch formation, this result suggests that caveolin-1 facilitates the prior action of GnT-III, relative to the other GnTs, on the nascent sugar chains in the Golgi apparatus and that GnT-III is redistributed in the earlier Golgi subcompartment by caveolin-1. Indeed, when caveolin-1 was expressed in human hepatoma cells, it was found to be co-localized with GnT-III, as evidenced by the fractionation of Triton X-100-insoluble cellular membranes by density Caveolin-1 the of sugar chains the of the intra-Golgi subcompartment localization of this In an investigation of the mechanism underlying the functional sublocalization of glycosyltransferases within the Golgi apparatus, caveolin-1 was identified as a possible cellular factor. Caveolin-1 appears to regulate the localization of N-acetylglucosaminyltransferase III (GnT-III) in the intra-Golgi subcompartment. Structural analyses of total cellular N-glycans indicated that the overexpression of GnT-III in human hepatoma cells, in which caveolin-1 is not expressed, failed to reduce branch formation, whereas expression of caveolin-1 led to a dramatic decrease in the extent of branching with no enhancement in GnT-III activity. Because the addition of a bisecting GlcNAc by GnT-III to the core β-Man in N-glycans prevents the action of GnT-IV and GnT-V, both of which are involved in branch formation, this result suggests that caveolin-1 facilitates the prior action of GnT-III, relative to the other GnTs, on the nascent sugar chains in the Golgi apparatus and that GnT-III is redistributed in the earlier Golgi subcompartment by caveolin-1. Indeed, when caveolin-1 was expressed in human hepatoma cells, it was found to be co-localized with GnT-III, as evidenced by the fractionation of Triton X-100-insoluble cellular membranes by density Caveolin-1 the of sugar chains the of the intra-Golgi subcompartment localization of this III (GnT-III) is of the glycosyltransferases that the of GlcNAc an to a core of a in the of a a bisecting GlcNAc a bisecting GlcNAc not as GnT-IV and GnT-V, which the of a an and a an that the action of was by the addition of a bisecting GlcNAc the the action of GnT-III prior to in the Golgi to an of the of Indeed, the expression of GnT-III an in in of in the of cells, the expression of GnT-III led to a dramatic decrease in by a in are in which a GnT-III not to a decrease in In the of human hepatoma cells, the overexpression of GnT-III in a dramatic in not a decrease of the of be that GnT-IV and prior to GnT-III in and cells, the of GnT-III, and are the in both of and GnT-IV and are to on the sugar a of are in the of GnT-III action both of cells, a mechanism be that is the prior action of GnT-III in as the of are by the action of the of action in by sublocalization within the Golgi that the and of the glycosyltransferases a in intra-Golgi functional localization the of in and hepatoma cells, that the functional localization of glycosyltransferases be by the is possible that this cellular other that caveolin-1 is a that a in the functional localization of GnT-III within the subcompartment of the Golgi apparatus, it is that a of caveolin-1 and that hepatoma not caveolin-1 in to expression in cells, is that caveolin-1 is not on the of the the Golgi apparatus glycosyltransferases it is that caveolin-1 as a the functional localization of this that GnT-III and and the a found that the expression of caveolin-1 to a dramatic decrease in the extent of branching of N-glycans of the addition of a bisecting a of GnT-III, the of GnT-III that caveolin-1 the functional localization of GnT-III, the mechanism that the intra-Golgi functional sublocalization of glycosyltransferases and the of in the the and and GlcNAc the and and by of human caveolin-1 was by human was with the and to the both was the of a expression a and the is to as of was by of the and in of with and a of and of with and an expression expression by to the in a by a with and by to the by to analyses and in and Triton was to to a by with was with the the was with the with an the by an of with and with to an with to as and as the was the of GnT-III and GnT-V, and the a of the of GnT-V, was the of of in the and as a was by and the was in a was by was a and the and the was with a and of and and of the cellular was by in and to cellular and and in of the and as by the by as by with the and the by on an with sugar chains with and sugar chains by a was a sugar chains as of Caveolin-1 a by in the of a with Triton and and on of cellular and by the was on the of a and in a the of the and the was by the addition of in of Triton X-100-insoluble of was in and the of with Triton on the with a to and with an of was with a and in a the of the and the total in was with in was as a in of of and and and by with was in a was in of a of and and to was on in Triton was in and to the of as a the of caveolin-1 on GnT-III action and on of human hepatoma cells, which not caveolin-1 of caveolin-1 was by an and not the other GnT-III GnT-III not be by an of expression In cells, which by with the GnT-III expression the GnT-III was that in cells, whereas the of other the In with the the GnT-III was to a in the the of with a caveolin-1 expression of cells, which as in GnT-III in the was with in the cells, and the of GnT-IV and of the expression of caveolin-1 of suggests that caveolin-1 not on the expression and of the functional of caveolin-1 expression on was analyses and in with which to to a with that of in which GnT-III is no in GnT-III activity. In the of the in the that in the that caveolin-1 of that are the of the of GnT-III, as found in the cells, the of bisecting GlcNAc are in both of by the no was the cells, and of the of by of and by to the and of total N-glycans the and with to core by of the and are in In the of cells, was the of GnT-IV and to of and Because and sugar chains not the the and not relative of the possible of the core are in overexpression of GnT-III the of to as with the in the of the with and relative to the total sugar chains that GnT-III GnT-IV and the other caveolin-1 expression led to an in the of the GnT-III was not In to the of GnT-III the of with the bisecting GlcNAc was In the cells, the was not and and of the sugar chains in earlier it that GnT-IV and are to on sugar chains in whereas GnT-III is of with of in the to that caveolin-1 the prior action of GnT-III on the nascent relative to other GnTs, which as in the Golgi is that caveolin-1 the subcompartment of GnT-III within the Golgi of the sugar chains and chains total cellular by and are with that the of the are the to the in are and in the GlcNAc and of of sugar of core of sugar chains are and the of and is total of is relative of total total and total are total of is relative of and with the bisecting GlcNAc are of total is it was found that caveolin-1 the in the of a mechanism underlying the of the by the GnT-III and caveolin-1 was expressed GnT-III the was the in the as in of this of GnT-III with caveolin-1 was not GnT-III was in to and of GnT-III and caveolin-1. GnT-III was expressed in and and with an by with and of GnT-III was indicated was as by and with the indicated a GnT-III was found to be with caveolin-1. a of GnT-III was with a not of the of GnT-III in the with the was that GnT-III is with caveolin-1 in the of GnT-III with caveolin-1 was cells, which caveolin-1. In the with GnT-III, the result was as in the not the that GnT-III is with caveolin-1 within are to be with membranes a both the and to on the the other the of GnT-III is in the and it be that GnT-III and caveolin-1 is to an it was that caveolin-1 the Golgi by and that GnT-III caveolin-1 in suggests the that GnT-III to caveolin-1 within the Golgi the of GnT-III and caveolin-1 and to the of GnT-III is with caveolin-1 GnT-III was expressed in in an to the of and with as a caveolin-1 was with the addition of Golgi GnT-III was to that are in a suggests that caveolin-1 and GnT-III are the Golgi the GnT-III and caveolin-1 is the Golgi fractionation by density of the Triton X-100-insoluble membranes was in in the of of cells, caveolin-1 was in the Triton X-100-insoluble the other in the of GnT-III, a of caveolin-1 was not the Triton X-100-insoluble membranes in the was found to be co-localized with that caveolin-1 is of a with GnT-III in the Golgi the other an of caveolin-1 the Golgi was not in cells, GnT-V, was in Golgi not the with caveolin-1 to be to of the of caveolin-1 with of caveolin-1 to Triton and the GnT-III with Triton and with a of by and with indicated of caveolin-1 to and the GnT-III with and on the of the be found of by and with the caveolin-1 the of a was In the of of cells, caveolin-1 was in the of the that it is in the result is with localization of caveolin-1 the as a as the other the overexpression of GnT-III in a of as indicated by to a it be that this GnT-III and caveolin-1 the prior action of GnT-III to GnT-IV and the of GnT-III an of the Golgi apparatus, and by the expression of that caveolin-1 with GnT-III in and that as a result expression in a of total cellular N-glycans that the is the sugar in the of cells, sugar chains are the in the both and caveolin-1 GnT-III the expression of caveolin-1 led to an to that found in it is that the of by caveolin-1 is to the of GnT-III overexpression failed to reduce branch in sugar chains be the bisecting GlcNAc a of GnT-III, is of and of branching the of in the in and suggests that GnT-IV and are the earlier subcompartment in the Golgi apparatus relative to the other the of the sugar in that caveolin-1 facilitates the prior action of GnT-III on nascent and it is that GnT-III be to an earlier subcompartment in the as the result of caveolin-1 the of be the relative of glycosyltransferases in a in cells, whereas not the in functional of which be the Golgi apparatus the as Golgi glycosyltransferases the of of other glycosyltransferases in of an in subcompartment the of glycosyltransferases as in the Golgi subcompartment. of be by the is that the cellular other glycosyltransferases are this and in the it is that caveolin-1 in the of GnT-III on are by the action of glycosyltransferases the to the the of action of which be with sublocalization in the as as expression and be a in the of cellular the of cellular which are involved in the functional localization of is an to the of glycosyltransferases and to the of III (GnT-III) is of the glycosyltransferases that the of GlcNAc an to a core of a in the of a a bisecting GlcNAc a bisecting GlcNAc not as GnT-IV and GnT-V, which the of a an and a an that the action of was by the addition of a bisecting GlcNAc the the action of GnT-III prior to in the Golgi to an of the of Indeed, the expression of GnT-III an in in of in the of cells, the expression of GnT-III led to a dramatic decrease in by a in are in which a GnT-III not to a decrease in In the of human hepatoma cells, the overexpression of GnT-III in a dramatic in not a decrease of the of be that GnT-IV and prior to GnT-III in and cells, the of GnT-III, and are the in both of and GnT-IV and are to on the sugar a of are in the of GnT-III action both of cells, a mechanism be that is the prior action of GnT-III in as the of are by the action of the of action in by sublocalization within the Golgi that the and of the glycosyltransferases a in intra-Golgi functional localization the of in and hepatoma cells, that the functional localization of glycosyltransferases be by the is possible that this cellular other that caveolin-1 is a that a in the functional localization of GnT-III within the subcompartment of the Golgi apparatus, it is that a of caveolin-1 and that hepatoma not caveolin-1 in to expression in cells, is that caveolin-1 is not on the of the the Golgi apparatus glycosyltransferases it is that caveolin-1 as a the functional localization of this that GnT-III and and the a found that the expression of caveolin-1 to a dramatic decrease in the extent of branching of N-glycans of the addition of a bisecting a of GnT-III, the of GnT-III that caveolin-1 the functional localization of GnT-III, the mechanism that the intra-Golgi functional sublocalization of glycosyltransferases and the of in the the and and GlcNAc the and and by of human caveolin-1 was by human was with the and to the both was the of a expression a and the is to as of was by of the and in of with and a of and of with and an expression expression by to the in a by a with and by to the by to analyses and in and Triton was to to a by with was with the the was with the with an the by an of with and with to an with to as and as the was the of GnT-III and GnT-V, and the a of the of GnT-V, was the of of in the and as a was by and the was in a was by was a and the and the was with a and of and and of the cellular was by in and to cellular and and in of the and as by the by as by with the and the by on an with sugar chains with and sugar chains by a was a sugar chains as of Caveolin-1 a by in the of a with Triton and and on of cellular and by the was on the of a and in a the of the and the was by the addition of in of Triton X-100-insoluble of was in and the of with Triton on the with a to and with an of was with a and in a the of the and the total in was with in was as a in of of and and and by with was in a was in of a of and and to was on in Triton was in and to the of as a the and and GlcNAc the and and by of human caveolin-1 was by human was with the and to the both was the of a expression a and the is to as of was by of the and in of with and a of and of with and an expression expression by to the in a by a with and by to the by to analyses and in and Triton was to to a by with was with the the was with the with an the by an of with and with to an with to as and as the was the of GnT-III and GnT-V, and the a of the of GnT-V, was the of of in the and as a was by and the was in a was by was a and the and the was with a and of and and of the cellular was by in and to cellular and and in of the and as by the by as by with the and the by on an with sugar chains with and sugar chains by a was a sugar chains as of Caveolin-1 a by in the of a with Triton and and on of cellular and by the was on the of a and in a the of the and the was by the addition of in of Triton X-100-insoluble of was in and the of with Triton on the with a to and with an of was with a and in a the of the and the total in was with in was as a in of of and and and by with was in a was in of a of and and to was on in Triton was in and to the of as a the of caveolin-1 on GnT-III action and on of human hepatoma cells, which not caveolin-1 of caveolin-1 was by an and not the other GnT-III GnT-III not be by an of expression In cells, which by with the GnT-III expression the GnT-III was that in cells, whereas the of other the In with the the GnT-III was to a in the the of with a caveolin-1 expression of cells, which as in GnT-III in the was with in the cells, and the of GnT-IV and of the expression of caveolin-1 of suggests that caveolin-1 not on the expression and of the functional of caveolin-1 expression on was analyses and in with which to to a with that of in which GnT-III is no in GnT-III activity. In the of the in the that in the that caveolin-1 of that are the of the of GnT-III, as found in the cells, the of bisecting GlcNAc are in both of by the no was the cells, and of total N-glycans the and with to core by of the and are in In the of cells, was the of GnT-IV and to of and Because and sugar chains not the the and not relative of the possible of the core are in overexpression of GnT-III the of to as with the in the of the with and relative to the total sugar chains that GnT-III GnT-IV and the other caveolin-1 expression led to an in the of the GnT-III was not In to the of GnT-III the of with the bisecting GlcNAc was In the cells, the was not and and of the sugar chains in earlier it that GnT-IV and are to on sugar chains in whereas GnT-III is of with of in the to that caveolin-1 the prior action of GnT-III on the nascent relative to other GnTs, which as in the Golgi is that caveolin-1 the subcompartment of GnT-III within the Golgi of the sugar chains and chains total cellular by and are with that the of the are the to the in are and in the GlcNAc and of of sugar of core of sugar chains are and the of and is total of is relative of total total and total are total of is relative of and with the bisecting GlcNAc are of total is it was found that caveolin-1 the in the of a mechanism underlying the of the by the GnT-III and caveolin-1 was expressed GnT-III the was the in the as in of this of GnT-III with caveolin-1 was not GnT-III was in to and of GnT-III and caveolin-1. GnT-III was expressed in and and with an by with and of GnT-III was indicated was as by and with the indicated a GnT-III was found to be with caveolin-1. a of GnT-III was with a not of the of GnT-III in the with the was that GnT-III is with caveolin-1 in the of GnT-III with caveolin-1 was cells, which caveolin-1. In the with GnT-III, the result was as in the not the that GnT-III is with caveolin-1 within are to be with membranes a both the and to on the the other the of GnT-III is in the and it be that GnT-III and caveolin-1 is to an it was that caveolin-1 the Golgi by and that GnT-III caveolin-1 in suggests the that GnT-III to caveolin-1 within the Golgi the of GnT-III and caveolin-1 and to the of GnT-III is with caveolin-1 GnT-III was expressed in in an to the of and with as a caveolin-1 was with the addition of Golgi GnT-III was to that are in a suggests that caveolin-1 and GnT-III are the Golgi the GnT-III and caveolin-1 is the Golgi fractionation by density of the Triton X-100-insoluble membranes was in in the of of cells, caveolin-1 was in the Triton X-100-insoluble the other in the of GnT-III, a of caveolin-1 was not the Triton X-100-insoluble membranes in the was found to be co-localized with that caveolin-1 is of a with GnT-III in the Golgi the other an of caveolin-1 the Golgi was not in cells, GnT-V, was in Golgi not the with caveolin-1 to be to of the of caveolin-1 with of caveolin-1 to Triton and the GnT-III with Triton and with a of by and with indicated of caveolin-1 to and the GnT-III with and on the of the be found of by and with the caveolin-1 the of a was In the of of cells, caveolin-1 was in the of the that it is in the result is with localization of caveolin-1 the as a as the other the overexpression of GnT-III in a of as indicated by to a it be that this GnT-III and caveolin-1 the prior action of GnT-III to GnT-IV and the of GnT-III an of the Golgi apparatus, and by the expression of caveolin-1. the of caveolin-1 on GnT-III action and on of human hepatoma cells, which not caveolin-1 of caveolin-1 was by an and not the other GnT-III GnT-III not be by an of expression In cells, which by with the GnT-III expression the GnT-III was that in cells, whereas the of other the In with the the GnT-III was to a in the the of with a caveolin-1 expression of cells, which as in GnT-III in the was with in the cells, and the of GnT-IV and of the expression of caveolin-1 of suggests that caveolin-1 not on the expression and of the functional of caveolin-1 expression on was analyses and in with which to to a with that of in which GnT-III is no in GnT-III activity. In the of the in the that in the that caveolin-1 of that are the of the of GnT-III, as found in the cells, the of bisecting GlcNAc are in both of by the no was the cells, and of total N-glycans the and with to core by of the and are in In the of cells, was the of GnT-IV and to of and Because and sugar chains not the the and not relative of the possible of the core are in overexpression of GnT-III the of to as with the in the of the with and relative to the total sugar chains that GnT-III GnT-IV and the other caveolin-1 expression led to an in the of the GnT-III was not In to the of GnT-III the of with the bisecting GlcNAc was In the cells, the was not and and of the sugar chains in earlier it that GnT-IV and are to on sugar chains in whereas GnT-III is of with of in the to that caveolin-1 the prior action of GnT-III on the nascent relative to other GnTs, which as in the Golgi is that caveolin-1 the subcompartment of GnT-III within the Golgi it was found that caveolin-1 the in the of a mechanism underlying the of the by the GnT-III and caveolin-1 was expressed GnT-III the was the in the as in of this of GnT-III with caveolin-1 was not GnT-III was in to a GnT-III was found to be with caveolin-1. a of GnT-III was with a not of the of GnT-III in the with the was that GnT-III is with caveolin-1 in the of GnT-III with caveolin-1 was cells, which caveolin-1. In the with GnT-III, the result was as in the not the that GnT-III is with caveolin-1 within are to be with membranes a both the and to on the the other the of GnT-III is in the and it be that GnT-III and caveolin-1 is to an it was that caveolin-1 the Golgi by and that GnT-III caveolin-1 in suggests the that GnT-III to caveolin-1 within the Golgi the of GnT-III and caveolin-1 and to the of GnT-III is with caveolin-1 GnT-III was expressed in in an to the of and with as a caveolin-1 was with the addition of Golgi GnT-III was to that are in a suggests that caveolin-1 and GnT-III are the Golgi the GnT-III and caveolin-1 is the Golgi fractionation by density of the Triton X-100-insoluble membranes was in in the of of cells, caveolin-1 was in the Triton X-100-insoluble the other in the of GnT-III, a of caveolin-1 was not the Triton X-100-insoluble membranes in the was found to be co-localized with that caveolin-1 is of a with GnT-III in the Golgi the other an of caveolin-1 the Golgi was not in cells, GnT-V, was in Golgi not the with caveolin-1 to be to the of a was In the of of cells, caveolin-1 was in the of the that it is in the result is with localization of caveolin-1 the as a as the other the overexpression of GnT-III in a of as indicated by to a it be that this GnT-III and caveolin-1 the prior action of GnT-III to GnT-IV and the of GnT-III an of the Golgi apparatus, and by the expression of caveolin-1. that caveolin-1 with GnT-III in and that as a result expression in a of total cellular N-glycans that the is the sugar in the of cells, sugar chains are the in the both and caveolin-1 GnT-III the expression of caveolin-1 led to an to that found in it is that the of by caveolin-1 is to the of GnT-III overexpression failed to reduce branch in sugar chains be the bisecting GlcNAc a of GnT-III, is of and of branching the of in the in and suggests that GnT-IV and are the earlier subcompartment in the Golgi apparatus relative to the other the of the sugar in that caveolin-1 facilitates the prior action of GnT-III on nascent and it is that GnT-III be to an earlier subcompartment in the as the result of caveolin-1 the of be the relative of glycosyltransferases in a in cells, whereas not the in functional of which be the Golgi apparatus the as Golgi glycosyltransferases the of of other glycosyltransferases in of an in subcompartment the of glycosyltransferases as in the Golgi subcompartment. of be by the is that the cellular other glycosyltransferases are this and in the it is that caveolin-1 in the of GnT-III on are by the action of glycosyltransferases the to the the of action of which be with sublocalization in the as as expression and be a in the of cellular the of cellular which are involved in the functional localization of is an to the of glycosyltransferases and to the of that caveolin-1 with GnT-III in and that as a result expression in a of total cellular N-glycans that the is the sugar in the of cells, sugar chains are the in the both and caveolin-1 GnT-III the expression of caveolin-1 led to an to that found in it is that the of by caveolin-1 is to the of GnT-III overexpression failed to reduce branch in sugar chains be the bisecting GlcNAc a of GnT-III, is of and of branching the of in the in and suggests that GnT-IV and are the earlier subcompartment in the Golgi apparatus relative to the other the of the sugar in that caveolin-1 facilitates the prior action of GnT-III on nascent and it is that GnT-III be to an earlier subcompartment in the as the result of caveolin-1 the of be the relative of glycosyltransferases in a in cells, whereas not the in functional of Because which be the Golgi apparatus the as Golgi glycosyltransferases the of of other glycosyltransferases in of an in subcompartment the of glycosyltransferases as in the Golgi subcompartment. of be by the is that the cellular other glycosyltransferases are this and in the it is that caveolin-1 in the of GnT-III on are by the action of glycosyltransferases the to the the of action of which be with sublocalization in the as as expression and be a in the of cellular the of cellular which are involved in the functional localization of is an to the of glycosyltransferases and to the of