Productive Folding of Tyrosinase Ectodomain Is Controlled by the Transmembrane Anchor

Abstract

Transmembrane domains (TMDs) are known as structural elements required for the insertion into the membrane of integral membrane proteins. We have provided here an example showing that the presence of the TMD is compulsory for the productive folding pathway of a membrane-anchored glycoprotein. Tyrosinase, a type I transmembrane protein whose insertion into the melanosomal membrane initiates melanin synthesis, is misfolded and degraded when expressed as a truncated polypeptide. We used constructs of tyrosinase ectodomain fused with chimeric TMDs or glycosylphosphatidylinositol anchor to gain insights into how the TMD enables the productive folding pathway of the ectodomain. We found that in contrast to the soluble constructs, the membrane-anchored chimeras fold into the native conformation, which allows their endoplasmic reticulum exit. They recruit calnexin to monitor their productive folding pathway the of calnexin the truncated is in an We that the transmembrane anchor of a protein the folding pathway of the ectodomain. Transmembrane domains (TMDs) are known as structural elements required for the insertion into the membrane of integral membrane proteins. We have provided here an example showing that the presence of the TMD is compulsory for the productive folding pathway of a membrane-anchored glycoprotein. Tyrosinase, a type I transmembrane protein whose insertion into the melanosomal membrane initiates melanin synthesis, is misfolded and degraded when expressed as a truncated polypeptide. We used constructs of tyrosinase ectodomain fused with chimeric TMDs or glycosylphosphatidylinositol anchor to gain insights into how the TMD enables the productive folding pathway of the ectodomain. We found that in contrast to the soluble constructs, the membrane-anchored chimeras fold into the native conformation, which allows their endoplasmic reticulum exit. They recruit calnexin to monitor their productive folding pathway the of calnexin the truncated is in an We that the transmembrane anchor of a protein the folding pathway of the ectodomain. of soluble and of when the is into the endoplasmic reticulum used endoplasmic soluble used endoplasmic soluble the and is an folding and in the presence of the calnexin and and the the ectodomain of a is the folding and of the transmembrane domains (TMDs) in the is known of the of the TMD in the folding of the protein ectodomain. the TMD a structural required to anchor the protein to the membrane or an in the folding the as a tyrosinase a type I membrane whose in the presence of the in and TMD in the and is to the are here to the initiates the melanin protein is a and an for the of the of tyrosinase for is for the of tyrosinase that are for the of that a truncated tyrosinase the TMD and is in the and and degraded the pathway how the TMD of tyrosinase the productive folding and of have in which the of tyrosinase a of TMDs and a glycosylphosphatidylinositol We found that soluble tyrosinase chimeras to the membrane a anchor or a of TMDs a and the folding is with calnexin which is for membrane-anchored of the TMD is to a calnexin that a productive folding pathway of and the of in of and of and with a is a and of and of for tyrosinase chimeras the as soluble tyrosinase with the of transmembrane domains the and in the and and the in with and in the of used to the of the in the of constructs, and of used in in a of the tyrosinase a the to a the to in in the and are in of and in in used to tyrosinase of of in the for with of for or and for the the in with and and for to the the of the in to the and as and and with the of of protein and for with in the for in with tyrosinase protein with and the protein in the of as with and the with with and with in and and with constructs the the with and with for or and with for in with the in for with with the in for with in and with a the for membrane in the folding and of soluble tyrosinase chimeras in which the ectodomain is to the membrane of membrane insertion and a anchor used a tyrosinase the ectodomain and the tyrosinase TMD the soluble truncated the and truncated the the tyrosinase TMD with the TMD of protein which is known to to the membrane in which the ectodomain to the TMD of the which to a in which the ectodomain fused with a TMD to and of the TMD is to tyrosinase in a a tyrosinase of the ectodomain and the TMD and the with the and tyrosinase and soluble tyrosinase in the used as in or in the that the tyrosinase the and the found in the tyrosinase and in soluble tyrosinase of tyrosinase is required for with in and the or with and to and with is of the the in with or and with and for as and is of the and the their the the of the and in an with or with and with in to the membrane in is a of the in the that tyrosinase to in in with a for membrane to the of the have an the of with the of and with the calnexin in and with and soluble tyrosinase with and tyrosinase with the with a of and a the the with calnexin to the to the a in the presence of which the of with the in a in the of calnexin with and calnexin in the presence of TMD calnexin of truncated with to the of and with with to calnexin and the with the of the the with to of the with and and and expressed as of the tyrosinase the of the in the presence of as in for the with calnexin used as a with as in for and as in for calnexin the and with and the of the the of the tyrosinase soluble truncated the the We found that to the that truncated in the of the expressed as a to with calnexin expressed a in the of the soluble soluble with calnexin and are in the the of the is for the tyrosinase ectodomain that the calnexin is the of the of that the of the tyrosinase TMD to the soluble ectodomain the with of the calnexin in the presence of a I and that the is the is required for tyrosinase the for to of and the of the with calnexin the of the ectodomain. the folding of and soluble tyrosinase the of to with or for for and and or with to found with to and and with the in and as a and with a in in and for of the the that folding in the of in and of and that are in and with for and for and with and and and presence of is the of the as for that the of the for with in as for that and the is of folding pathway folding is in a in a is when the with a and a is a in the of the in the of synthesis, which the of of the tyrosinase is the of the in contrast to with a that the the is the of the are to have the in and the the with a in and in to tyrosinase with with the as the tyrosinase and with the the soluble a folding pathway the of in which the are the of the protein is into an folding pathway of the that are that are the protein into the native soluble tyrosinase is a protein with a to of the the in of and have that in the of the the ectodomain of tyrosinase is to a of of the TMD that in the of the TMD the ectodomain is to with to folding that in an the of the and of the transmembrane anchor the of the tyrosinase ectodomain tyrosinase chimeras with the of tyrosinase and the of or of the the is in the the constructs are of of that with the of the the TMD of the protein the the as that the membrane in TMD is for tyrosinase and calnexin with in the or with in and to with is of the for the membrane and of and the of to the of and with with to and the with the of the the with to of the with and and and expressed as a of the tyrosinase the of calnexin of the in of to of and of the calnexin with for the with the with the that the of calnexin with tyrosinase is of the TMD with the in showing that the is in the of the that the of the TMD the of the tyrosinase the of with the anchor is a for the calnexin that the the productive of the tyrosinase ectodomain. the anchor to the tyrosinase ectodomain of the of the of the to the membrane in a as in the of a anchor the calnexin and the of the tyrosinase ectodomain. with in or with in and to with is of the for of the protein membrane in to the of with with to and the with the of the the with to the of the and expressed as of the tyrosinase the of of with calnexin in that the presence of a anchor to the of the ectodomain with calnexin with a of and that a anchor for the TMD and is of the of the tyrosinase ectodomain with calnexin with and as tyrosinase and have used and chimeric tyrosinase to the of the for the folding pathway of the tyrosinase ectodomain. We that the tyrosinase folding pathway and that the of the is the in the of the the ectodomain is in a that and to that is a the calnexin and the of in tyrosinase that the in a productive folding tyrosinase into a the the native conformation, the presence of that in the of or tyrosinase a folding pathway the truncated is in the the of the of native a the the the membrane in the of or in the for soluble the of the folding the of the tyrosinase that tyrosinase calnexin and the soluble tyrosinase the calnexin the of and productive folding the presence of a TMD is a for the of with calnexin is for the soluble the with is in the of the a misfolded protein with calnexin with the an to the TMD is required for the We found that TMDs the with that is the TMD of tyrosinase and the of found in the presence of an of the TMD to tyrosinase ectodomain calnexin with the ectodomain and folding and of the the of the TMD with a anchor to the calnexin and the that a membrane anchor is required to the protein into the of the anchor is to the the that the membrane the calnexin in a the folding showing a of the folding of transmembrane folding for the folding of the transmembrane the with a folding that is to the folding is calnexin with and the of TMD the calnexin is to the calnexin to a of that in the of is with a showing that the folding pathway in provided that the of folding in the are for which the is to a to and in is a protein that with and with the for TMD calnexin to the folding into the native the to the of the and which in the of the TMD and the transmembrane whose TMDs are the the native folding of the ectodomain that the TMD of the membrane-anchored tyrosinase is compulsory for productive folding the soluble constructs, the membrane-anchored tyrosinase chimeras a productive folding pathway that allows their TMD tyrosinase folding pathway calnexin the of the the is a for the TMD in the folding of that to of the of TMDs and of the of protein the and the folding is the of for transmembrane that the of of the tyrosinase to to the for and as a of soluble and of when the is into the endoplasmic reticulum used endoplasmic soluble used endoplasmic soluble the and is an folding and in the presence of the calnexin and and the the ectodomain of a is the folding and of the transmembrane domains (TMDs) in the is known of the of the TMD in the folding of the protein ectodomain. the TMD a structural required to anchor the protein to the membrane or an in the folding the as a tyrosinase a type I membrane whose in the presence of the in and TMD in the and is to the are here to the initiates the melanin protein is a and an for the of the of tyrosinase for is for the of tyrosinase that are for the of that a truncated tyrosinase the TMD and is in the and and degraded the pathway how the TMD of tyrosinase the productive folding and of have in which the of tyrosinase a of TMDs and a glycosylphosphatidylinositol We found that soluble tyrosinase chimeras to the membrane a anchor or a of TMDs a and the folding is with calnexin which is for membrane-anchored of the TMD is to a calnexin that a productive folding pathway of and the of in of and of and with a is a and of and of for tyrosinase chimeras the as soluble tyrosinase with the of transmembrane domains the and in the and and the in with and in the of used to the of the in the of constructs, and of used in in a of the tyrosinase a the to a the to in in the and are in of and in in used to tyrosinase of of in the for with of for or and for the the in with and and for to the the of the in to the and as and and with the of of protein and for with in the for in with tyrosinase protein with and the protein in the of as with and the with with and with in and and with constructs the the with and with for or and with for in with the in for with with the in for with in and with a and the of in of and of and with a is a and of and of for tyrosinase chimeras the as soluble tyrosinase with the of transmembrane domains the and in the and and the in with and in the of used to the of the in the of constructs, and of the tyrosinase a the to a the to in in the and are in of and in in used to tyrosinase of of in the for with of for or and for the the in with and and for to the the of the in to the and as and and with the of of protein and for with in the for in with tyrosinase protein with and the protein in the of as with and the with with and with in and and with constructs the the with and with for or and with for in with the in for with with the in for with in and with a the for membrane in the folding and of soluble tyrosinase chimeras in which the ectodomain is to the membrane of membrane insertion and a anchor used a tyrosinase the ectodomain and the tyrosinase TMD the soluble truncated the and truncated the the tyrosinase TMD with the TMD of protein which is known to to the membrane in which the ectodomain to the TMD of the which to a in which the ectodomain fused with a TMD to and of the TMD is to tyrosinase in a a tyrosinase of the ectodomain and the TMD and the with the and tyrosinase and soluble tyrosinase in the used as in or in the that the tyrosinase the and the found in the tyrosinase and in soluble tyrosinase their the the of the and in an with or with and with in to the membrane in is a of the in the that tyrosinase to in in with a for membrane to the of the have an the of with the of and with the calnexin in and with and soluble tyrosinase with and tyrosinase with the with a of and a the the with calnexin to the to the a in the presence of which the of with the in a in the of calnexin with and calnexin in the presence of TMD calnexin of truncated with to the of and with with to calnexin and the with the of the the with to of the with and and and expressed as of the tyrosinase the of the in the presence of as in for the with calnexin used as a with as in for and as in for calnexin the and with and the of the the of the tyrosinase soluble truncated the the We found that to the that truncated in the of the expressed as a to with calnexin expressed a in the of the soluble soluble with calnexin and are in the the of the is for the tyrosinase ectodomain that the calnexin is the of the of that the of the tyrosinase TMD to the soluble ectodomain the with of the calnexin in the presence of a I and that the is the is required for tyrosinase the for to of and the of the with calnexin the of the ectodomain. the folding of and soluble tyrosinase the of to with or for for and and or with to found with to and and with the in and as a and with a in in and for of the the that folding in the of in and of and that are in and with for and for and with and and and presence of is the of the as for that the of the for with in as for that and the is of folding pathway folding is in a in a is when the with a and a is a in the of the in the of synthesis, which the of of the tyrosinase is the of the in contrast to with a that the the is the of the are to have the in and the the with a in and in to tyrosinase with with the as the tyrosinase and with the the soluble a folding pathway the of in which the are the of the protein is into an folding pathway of the that are that are the protein into the native soluble tyrosinase is a protein with a to of the the in of and have that in the of the the ectodomain of tyrosinase is to a of of the TMD that in the of the TMD the ectodomain is to with to folding that in an the of the and of the transmembrane anchor the of the tyrosinase ectodomain tyrosinase chimeras with the of tyrosinase and the of or of the the is in the the constructs are of of that with the of the the TMD of the protein the the as that the membrane in TMD is for tyrosinase and calnexin with in the or with in and to with is of the for the membrane and of and the of to the of and with with to and the with the of the the with to of the with and and and expressed as a of the tyrosinase the of calnexin of the in of to of and of the calnexin with for the with the with the that the of calnexin with tyrosinase is of the TMD with the in showing that the is in the of the that the of the TMD the of the tyrosinase the of with the anchor is a for the calnexin that the the productive of the tyrosinase ectodomain. the anchor to the tyrosinase ectodomain of the of the of the to the membrane in a as in the of a anchor the calnexin and the of the tyrosinase ectodomain. with in or with in and to with is of the for of the protein membrane in to the of with with to and the with the of the the with to the of the and expressed as of the tyrosinase the of of with calnexin in that the presence of a anchor to the of the ectodomain with calnexin with a of and that a anchor for the TMD and is of the of the tyrosinase ectodomain with calnexin with and as tyrosinase and the for membrane in the folding and of soluble tyrosinase chimeras in which the ectodomain is to the membrane of membrane insertion and a anchor used a tyrosinase the ectodomain and the tyrosinase TMD the soluble truncated the and truncated the the tyrosinase TMD with the TMD of protein which is known to to the membrane in which the ectodomain to the TMD of the which to a in which the ectodomain fused with a TMD to and of the TMD is to tyrosinase in a a tyrosinase of the ectodomain and the TMD and the with the and tyrosinase and soluble tyrosinase in the used as in or in the that the tyrosinase the and the found in the tyrosinase and in soluble tyrosinase their the the of the and in an with or with and with in to the membrane in is a of the in the that tyrosinase to in in with a for membrane to the of the have an the of with the of and with the calnexin in and with and soluble tyrosinase with and tyrosinase with the with a of and a the the with calnexin to the to the a in the presence of which the of with the in a in the of calnexin with and calnexin in the presence of the of the the of the tyrosinase soluble truncated the the We found that to the that truncated in the of the expressed as a to with calnexin expressed a in the of the soluble soluble with calnexin and are in the the of the is for the tyrosinase ectodomain that the calnexin is the of the of that the of the tyrosinase TMD to the soluble ectodomain the with of the calnexin in the presence of a I and that the is the is required for tyrosinase the for to of and the of the with calnexin the of the ectodomain. the folding of and soluble tyrosinase the of to with or for for and and or with to found with to and and with the in and as a and with a in in and for of the the that folding in the of in and of and that are in in a is when the with a and a is a in the of the in the of synthesis, which the of of the tyrosinase is the of the in contrast to with a that the the is the of the are to have the in and the the with a in and in to tyrosinase with with the as the tyrosinase and with the the soluble a folding pathway the of in which the are the of the protein is into an folding pathway of the that are that are the protein into the native soluble tyrosinase is a protein with a to of the the in of and have that in the of the the ectodomain of tyrosinase is to a of of the TMD that in the of the TMD the ectodomain is to with to folding that in an the of the and of the transmembrane anchor the of the tyrosinase ectodomain tyrosinase chimeras with the of tyrosinase and the of or of the the is in the the constructs are of of that with the of the the TMD of the protein the the as that the membrane in calnexin of the in of to of and of the calnexin with for the with the with the that the of calnexin with tyrosinase is of the TMD with the in showing that the is in the of the that the of the TMD the of the tyrosinase ectodomain. the of with the anchor is a for the calnexin that the the productive of the tyrosinase ectodomain. the anchor to the tyrosinase ectodomain of the of the of the to the membrane in a as in the of with calnexin in that the presence of a anchor to the of the ectodomain with calnexin with a of and that a anchor for the TMD and is of the of the tyrosinase ectodomain with calnexin with and as tyrosinase and have used and chimeric tyrosinase to the of the for the folding pathway of the tyrosinase ectodomain. We that the tyrosinase folding pathway and that the of the is the in the of the the ectodomain is in a that and to that is a the calnexin and the of in tyrosinase that the in a productive folding tyrosinase into a the the native conformation, the presence of that in the of or tyrosinase a folding pathway the truncated is in the the of the of native a the the the membrane in the of or in the for soluble the of the folding the of the tyrosinase that tyrosinase calnexin and the soluble tyrosinase the calnexin the of and productive folding the presence of a TMD is a for the of with calnexin is for the soluble the with is in the of the a misfolded protein with calnexin with the an to the TMD is required for the We found that TMDs the with that is the TMD of tyrosinase and the of found in the presence of an of the TMD to tyrosinase ectodomain calnexin with the ectodomain and folding and of the the of the TMD with a anchor to the calnexin and the that a membrane anchor is required to the protein into the of the anchor is to the the that the membrane the calnexin in a the folding showing a of the folding of transmembrane folding for the folding of the transmembrane the with a folding that is to the folding is calnexin with and the of TMD the calnexin is to the calnexin to a of that in the of is with a showing that the folding pathway in provided that the of folding in the are for which the is to a to and in is a protein that with and with the for TMD calnexin to the folding into the native the to the of the and which in the of the TMD and the transmembrane whose TMDs are the the native folding of the ectodomain that the TMD of the membrane-anchored tyrosinase is compulsory for productive folding the soluble constructs, the membrane-anchored tyrosinase chimeras a productive folding pathway that allows their TMD tyrosinase folding pathway calnexin the of the the is a for the TMD in the folding of that to of the of TMDs and of the of protein the and the folding is the of for transmembrane that the of of the tyrosinase to to the for and as a We have used and chimeric tyrosinase to the of the for the folding pathway of the tyrosinase ectodomain. We that the tyrosinase folding pathway and that the of the is the in the of the the ectodomain is in a that and to that is a the calnexin and the of in tyrosinase that the in a productive folding tyrosinase into a the the native conformation, the presence of that in the of or tyrosinase a folding pathway the truncated is in the the of the of native a the the the membrane in the of or in the for soluble the of the folding the of the tyrosinase that tyrosinase calnexin and the soluble tyrosinase the calnexin the of and productive folding the presence of a TMD is a for the of with calnexin is for the soluble the with is in the of the a misfolded protein with calnexin with the an to the TMD is required for the We found that TMDs the with that is the TMD of tyrosinase and the of found in the presence of an of the TMD to tyrosinase ectodomain calnexin with the ectodomain and folding and of the the of the TMD with a anchor to the calnexin and the that a membrane anchor is required to the protein into the of the anchor is to the the that the membrane the calnexin in a the folding showing a of the folding of transmembrane folding for the folding of the transmembrane the with a folding that is to the folding is calnexin with and the of TMD the calnexin is to the calnexin to a of that in the of is with a showing that the folding pathway in provided that the of folding in the are for which the is to a to and in is a protein that with and with the for TMD calnexin to the folding into the native the to the of the and which in the of the TMD and the transmembrane whose TMDs are the the native folding of the ectodomain We that the TMD of the membrane-anchored tyrosinase is compulsory for productive folding the soluble constructs, the membrane-anchored tyrosinase chimeras a productive folding pathway that allows their TMD tyrosinase folding pathway calnexin the of the the is a for the TMD in the folding of that to of the of TMDs and of the of protein the and the folding is the of for transmembrane that the of of the tyrosinase to to the for and as a We for for the for the and for the