Phosphorylation Region of the Yeast Plasma-membrane H+-ATPase

Abstract

Mutations at the phosphorylation site (Asp-378) of the yeast plasma-membrane H+-ATPase have been shown previously to cause misfolding of the ATPase, preventing normal movement along the secretory pathway; Asp-378 mutations also block the biogenesis of co-expressed wild-type ATPase and lead to a dominant lethal phenotype. To ask whether these defects are specific for Asp-378 or whether the phosphorylation region as a whole is involved, alanine-scanning mutagenesis has been carried out to examine the role of 11 conserved residues flanking Asp-378. In the sec6–4 expression system (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940–7949), the mutant ATPases displayed varying abilities to reach the secretory vesicles that deliver plasma-membrane proteins to the cell surface. Indirect immunofluorescence of intact cells also gave evidence for a spectrum of behavior, ranging from mutant ATPases completely arrested (D378A, K379A, T380A, and T384A) or partially arrested in the endoplasmic reticulum to those that reached the plasma membrane in normal amounts (C376A, S377A, and G381A). Although the extent of ER retention varied among the mutants, the endoplasmic reticulum appeared to be the only secretory compartment in which the mutant ATPases accumulated. All of the mutant proteins that localized either partially or fully to the ER were also malfolded based on their abnormal sensitivity to trypsin. Among them, the severely affected mutants had a dominant lethal phenotype, and even the intermediate mutants caused a visible slowing of growth when co-expressed with wild-type ATPase. The effects on growth could be traced to the trapping of the wild-type enzyme with the mutant enzyme in the ER, as visualized by double label immunofluorescence. Taken together, the results indicate that the residues surrounding Asp-378 are critically important for ATPase maturation and transport to the cell surface. Mutations at the phosphorylation site (Asp-378) of the yeast plasma-membrane H+-ATPase have been shown previously to cause misfolding of the ATPase, preventing normal movement along the secretory pathway; Asp-378 mutations also block the biogenesis of co-expressed wild-type ATPase and lead to a dominant lethal phenotype. To ask whether these defects are specific for Asp-378 or whether the phosphorylation region as a whole is involved, alanine-scanning mutagenesis has been carried out to examine the role of 11 conserved residues flanking Asp-378. In the sec6–4 expression system (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940–7949), the mutant ATPases displayed varying abilities to reach the secretory vesicles that deliver plasma-membrane proteins to the cell surface. Indirect immunofluorescence of intact cells also gave evidence for a spectrum of behavior, ranging from mutant ATPases completely arrested (D378A, K379A, T380A, and T384A) or partially arrested in the endoplasmic reticulum to those that reached the plasma membrane in normal amounts (C376A, S377A, and G381A). Although the extent of ER retention varied among the mutants, the endoplasmic reticulum appeared to be the only secretory compartment in which the mutant ATPases accumulated. All of the mutant proteins that localized either partially or fully to the ER were also malfolded based on their abnormal sensitivity to trypsin. Among them, the severely affected mutants had a dominant lethal phenotype, and even the intermediate mutants caused a visible slowing of growth when co-expressed with wild-type ATPase. The effects on growth could be traced to the trapping of the wild-type enzyme with the mutant enzyme in the ER, as visualized by double label immunofluorescence. Taken together, the results indicate that the residues surrounding Asp-378 are critically important for ATPase maturation and transport to the cell surface. In eukaryotic cells, the first step in the biogenesis of plasma membrane proteins takes place in the endoplasmic reticulum, where there is an elaborate machinery to ensure correct protein folding and integration into the lipid bilayer (1Gething M.-J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3590) Google Scholar, 2Hammond C. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (587) Google Scholar). In recent years, several genetic diseases have been traced to mutations that damage the ability of particular proteins to interact properly with this biosynthetic machinery (3Brooks D.A. FEBS Lett. 1997; 409: 115-120Crossref PubMed Scopus (50) Google Scholar). Perhaps the best known example is cystic fibrosis, in which the deletion of a single phenylalanine residue (Phe-508) from a plasma-membrane chloride channel known as CFTR 1The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorERendoplasmic reticulumHAinfluenza hemagglutininPAGEpolyacrylamide gel electrophoresisFITCfluorescein isothiocyanate.1The abbreviations used are: CFTRcystic fibrosis transmembrane conductance regulatorERendoplasmic reticulumHAinfluenza hemagglutininPAGEpolyacrylamide gel electrophoresisFITCfluorescein isothiocyanate. leads to defective folding of the channel protein and retention in the ER (4Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1416) Google Scholar, 5Riordan J.R. Annu. Rev. Physiol. 1993; 55: 609-630Crossref PubMed Scopus (306) Google Scholar, 6Welsh M.J. Smith A.E. Cell. 1993; 73: 1251-1254Abstract Full Text PDF PubMed Scopus (1224) Google Scholar). Similarly, familial hypercholesterolemia, a disease characterized by elevated plasma low density lipoprotein cholesterol and an increased risk of premature coronary artery disease, can arise from amino acid substitutions that cause ER retention of the low density lipoprotein receptor (7Leitersdorf E. Hobbs H.H. Fourie A.M. Jacobs M. van der Westhyzen D.R. Coetzee G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7912-7916Crossref PubMed Scopus (37) Google Scholar, 8Djordjevic J.T. Bierli S. Smith R. Kroon P.A. Eur. J. Biochem. 1996; 239: 214-219Crossref PubMed Scopus (19) Google Scholar, 9Jensen H.K. Holst H. Jensen L.G. Jørgensen M.M. Andreasen P.H. Jensen T.G. Andresen B.S. Heath F. Hansen P.S. Neve S. Kristiansen K. Færgeman O. Kølvraa S. Bolund L. Gregersen N. Atherosclerosis. 1997; 131: 67-72Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). cystic fibrosis transmembrane conductance regulator endoplasmic reticulum influenza hemagglutinin polyacrylamide gel electrophoresis fluorescein isothiocyanate. cystic fibrosis transmembrane conductance regulator endoplasmic reticulum influenza hemagglutinin polyacrylamide gel electrophoresis fluorescein isothiocyanate. These observations make it important to understand the factors that contribute to the proper folding of plasma membrane proteins as well as the quality control mechanisms that prevent malfolded proteins from being transported to the cell surface. The budding yeastSaccharomyces cerevisiae can serve as a good model organism for such studies because of the increasingly detailed knowledge of its secretory system (10Schekman R. Curr. Opin. Cell Biol. 1992; 4: 587-592Crossref PubMed Scopus (114) Google Scholar). In the work to be described below, we have focused on the yeast plasma membrane H+-ATPase, which is encoded by the PMA1 gene and comprises ∼10% of total plasma-membrane protein (11Serrano R. Kielland-Brandt M.C. Fink G.R. Nature. 1986; 319: 689-693Crossref PubMed Scopus (572) Google Scholar, 12Rao R. Slayman C.W. Brambl R. Marzluf G.A. The Mycota. 3. Springer-Verlag, Berlin1996: 29-56Google Scholar). Based on hydropathy plots and trypsinolysis studies, the ATPase is thought to contain 10 transmembrane segments, four at the N-terminal end and six at the C-terminal end of the the and are at the of the membrane Slayman C.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with CFTR J.R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google and the low density lipoprotein receptor H.K. Holst H. Jensen L.G. Jørgensen M.M. Andreasen P.H. Jensen T.G. Andresen B.S. Heath F. Hansen P.S. Neve S. Kristiansen K. Færgeman O. Kølvraa S. Bolund L. Gregersen N. Atherosclerosis. 1997; 131: 67-72Abstract Full Text Full Text PDF PubMed Scopus (24) Google folding of the yeast PMA1 ATPase can be by a single amino acid first studies of a residue conserved in Asp-378 the of the and is by to a it an role in the of Asp-378 by or to cause of the mutant the biogenesis of co-expressed wild-type ATPase and to a dominant lethal R. S. Slayman C.W. N. Acad. Sci. 1992; PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, F. FEBS Lett. 1997; PubMed Scopus Google Scholar). studies have shown the protein to be into the ER lipid it is and severely in S. A. R. Slayman C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Asp-378 is a in it could a role in folding and the the region surrounding the phosphorylation site could be for folding and the secretory to the and role of residues the phosphorylation region of the ATPase. this in we have an of 11 conserved amino that Asp-378. of the mutant proteins has been for and on of wild-type ATPase. The results indicate that of the which at the site of a cause defects in ER retention and dominant that the region surrounding is critically important for proper folding and with the secretory In results that the quality control step for the ATPase is in the secretory ATPases in the ER, the ER quality control to the plasma membrane in the by is with and described previously by and R. Slayman C.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In this the of the PMA1 gene has been control of the Smith E. A. PubMed Scopus Google Scholar). also the sec6–4 that the step of the secretory the of secretory vesicles with the plasma with the ATPase to be arrested in the secretory vesicles for R. Slayman C.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). in the ATPase biogenesis studies, of PMA1 were either a acid or a acid the of the ATPase. F. N. K. E. and C. W. Slayman, in shown in the and were control of the in and The H. K. A. J. PubMed Google used to the into cells to the of the ATPase to the plasma The were also control in These were into cells to the of ATPase used in this from A. 1988; PubMed Scopus Google from A. 1988; PubMed Scopus Google from A. 1988; PubMed Scopus Google from A. 1988; PubMed Scopus Google in a The used to mutations into a of as described previously R. Slayman C.W. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). To the mutant and place or the mutations were into the expression described The of the mutations in the by of carried out to the expression of wild-type and mutant ATPases in total cells with either the wild-type or a mutant of were on at to for and to for R. Slayman C.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and of the cells, total were by S. A. R. Slayman C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of 10 of protein with in to to and with and by with and ATPase were visualized by immunofluorescence a of the of K. C. J. Cell Biol. PubMed Scopus Google Scholar). these cells were with or wild-type or PMA1 in with The were at in F. in and and which used as a to prevent in the the cells were to with for to expression of the ATPase were with and by as described previously K. C. J. Cell Biol. PubMed Scopus Google Scholar). were for immunofluorescence as described by K. C. J. Cell Biol. PubMed Scopus Google with the of a step in in which the and were by normal were from by H. by R. A. K. J. R. J. Cell Biol. PubMed Scopus Google and by M. Cell. Full Text PDF PubMed Scopus Google and as and were double were the and were the To control for of the and cells were and with of were with and in cells were with a and cells were on a channel for of and the to an of The of by the and the of by that the when with were with and by of and vesicles were from cells with the wild-type or a mutant a A. Slayman C.W. Biochem. 1997; PubMed Scopus Google Scholar). of ATPase and protein were carried out as described previously F. FEBS Lett. 1997; PubMed Scopus Google Scholar, A. Slayman C.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google as were trypsinolysis S. A. R. Slayman C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To examine the genetic of the cells with wild-type or mutant were on and were in and for growth on and either gene or mutant and wild-type The were at for and described a of this work to in the phosphorylation region of surrounding the site (Asp-378) at which the intermediate is To residues for were from ATPases in and from shown in there is a of from Asp-378 the acid is conserved in known ATPase. either of this there are residues and and that are in the and the from of there is at which is in known and is by or in the and at which is by or in by or in of the the J. J. Biol. PubMed Scopus Google a in the phosphorylation the at to and for a is by that are in Based on the of we carried out alanine-scanning mutagenesis from to it that at of the substitutions be lethal R. S. Slayman C.W. N. Acad. Sci. 1992; PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, F. FEBS Lett. 1997; PubMed Scopus Google Scholar, R. Slayman C.W. J. Biol. Chem. 1993; Full Text PDF PubMed Google a expression system R. Slayman C.W. J. Biol. Chem. Full Text PDF PubMed Google used for the of from the wild-type ATPase on the and by to a mutant on a and by a the a block in the step of the secretory to the of secretory vesicles in the to the vesicles and by with for mutant protein to the plasma shown in the expression of and were normal when with the wild-type expression in the of the and the severely with or ATPase in the secretory vesicles with an control In a total were and by as shown in In this of the mutant proteins could be at normal the and the there in either the or the of the mutant as for substitutions at Asp-378 R. S. Slayman C.W. N. Acad. Sci. 1992; PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, F. FEBS Lett. 1997; PubMed Scopus Google Scholar, R. Slayman C.W. J. Biol. Chem. 1993; Full Text PDF PubMed Google the of of the mutants to reach the secretory vesicles a in their to the cell of mutations in the phosphorylation region on expression and are as a of the of wild-type protein of secretory as by are the of to with an of at in the of 10 and as described are the of to with an of is as of were for mutant in which expression were of were for mutant in which expression were of are from ATPases and and are from ATPases and and are from ATPases and and were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of were for mutant in which expression were of are as a of the of wild-type protein of secretory as by are the of to with an of at in the of 10 and as described are the of to with an of is as of All are from ATPases and and Slayman C.W. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google were for mutant in which expression were of in a also severely affected in of the mutants Among those only displayed were in S377A, K379A, and The mutants had that were To examine the defects in a expression with immunofluorescence to the of the mutant In this mutant control on a and into yeast which a wild-type of PMA1 on the of the mutant protein by growth on and the cells were by immunofluorescence the mutants displayed a of In S377A, and there a of the plasma membrane by and ATPase this also the for the wild-type in and the ATPase to varying the plasma membrane and there were mutants (D378A, K379A, T380A, and T384A) in which the ATPase appeared only in these cells, of the plasma membrane that the were at in from the endoplasmic The yeast ER the with to the cell J. C. 7: PubMed Scopus Google Scholar). the expression of abnormal it can into R. M. L. J. J. Cell Biol. 1988; that are by the ER A. A. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, M. 1993; PubMed Scopus Google Scholar, R. Eur. J. Biochem. 1996; 239: PubMed Scopus Google Scholar). S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and F. FEBS Lett. 1997; PubMed Scopus Google have such in cells mutant of PMA1 ATPase, and an important of the to whether or of the of mutant ATPases in these and at the whether there is also in such as the To these immunofluorescence and were carried out on cells mutant PMA1 ATPases of the and the severely affected In to which the mutant as a for the ER Cell. Full Text PDF PubMed Scopus Google and for the A. K. J. R. J. Cell Biol. PubMed Scopus Google Scholar). In the of intermediate of the ATPase reached the plasma membrane of it with in the region and in of those described by S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google the of and on the In the cells that were in the even when amounts of were in the plasma membrane and ER and there to be a slowing of the secretory pathway; ATPase that to the ER could the to the cell surface. observations were for of the intermediate mutants and In severely affected mutants such as there evidence of ATPase at the plasma membrane or in the to the In we have shown that ATPase is into the lipid bilayer is with increased sensitivity to S. A. R. Slayman C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google it is the folding that the ATPase to be in the ER R. S. Slayman C.W. N. Acad. Sci. 1992; PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, F. FEBS Lett. 1997; PubMed Scopus Google Scholar). described the evidence for a spectrum of in mutants the phosphorylation region of the ATPase. ER in from to of the protein can to the secretory vesicles and to the cell surface. to whether the of the with the extent of the protein folding as by this of total of the mutant ATPases were with and the were by with ATPase and by and the mutants varied in their sensitivity to as shown in and were from the wild-type of at a of there were amounts of ATPase and a and displayed intermediate of and were in K379A, T380A, and T384A) were to as as of the and had been completely and were by there a protein folding and retention of the mutant the be described mutations at the phosphorylation site (Asp-378) of PMA1 have a dominant are lethal when co-expressed with the wild-type R. S. Slayman C.W. N. Acad. Sci. 1992; PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, F. FEBS Lett. 1997; PubMed Scopus Google Scholar). of to the genetic of mutations the phosphorylation this the cells used for the immunofluorescence of and were on or to or expression of the mutant shown in the mutations a of effects on cell K379A, T380A, and displayed a dominant phenotype, with growth on and had an intermediate slowing completely preventing growth on and and were on as well as the wild-type and with which be mutant ATPases with defects were either partially or completely dominant in the growth To as the co-expressed mutant and wild-type ATPases were in the mutant and wild-type were and the on and into example of this a mutant with an intermediate block in biogenesis is shown in cells and wild-type were to and by there of mutant and wild-type ATPases in and as in the and of the this with the results of we that the of the ATPase were arrested in the In when mutants such as were co-expressed with the ATPases reached the cell described in the the for this the that mutations at Asp-378 cause misfolding of the yeast PMA1 ATPase, to retention in the ER and dominant R. S. Slayman C.W. N. Acad. Sci. 1992; PubMed Scopus Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, F. FEBS Lett. 1997; PubMed Scopus Google Scholar). of the work to whether these are specific to Asp-378 or whether are by residues the phosphorylation is that the is the of 11 substitutions in the and caused defects in folding and in of dominant have been in only out of mutants in the and of PMA1 A. Slayman C.W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Based on these the surrounding Asp-378 a role in ATPase maturation and transport to the cell surface. at in mutants in the phosphorylation region can be along a based on their biogenesis of the mutant proteins (C376A, S377A, along the secretory as by either of of ATPase that has reached the secretory vesicles the sec6–4 expression R. Slayman C.W. J. Biol. Chem. Full Text PDF PubMed Google or immunofluorescence of ATPase in the plasma membrane of intact cells and expression in these mutant ATPases to be either (C376A, or well when by is out that of the mutants, and are of in secretory were only of the wild-type control for that of the ATPase to the secretory vesicles transport into the of the secretory of the effects of mutations in the phosphorylation in secretory shown by in shown by immunofluorescence. ER, endoplasmic plasma shown by by in secretory by a of dominant in which the mutant ATPase co-expressed with the wild-type could be because the mutant ATPase in the could be because the mutant ATPase in the could be because the mutant ATPase in the could be because the mutant ATPase in the could be because the mutant ATPase in the could be because the mutant ATPase in the shown by shown by immunofluorescence. ER, endoplasmic plasma shown by by in secretory by a of dominant in which the mutant ATPase co-expressed with the wild-type could be because the mutant ATPase in the in a the end of the mutants and T384A) with in were from secretory and were only in immunofluorescence of intact Based on their sensitivity to these mutant ATPases to be it that are by a quality control at the step of the secretory The six mutants were intermediate in their behavior, a of abilities and to reach the secretory vesicles in the sec6–4 of also intermediate based on immunofluorescence ATPase reached the cell of it in In the mutants and the ATPase appeared to be arrested in the of this intermediate in its sensitivity to and the were to in protein is that ATPase biogenesis is to substitutions in the phosphorylation studies have shown that the of residues in this of the protein is critically the of a single at and leads to biosynthetic of the ATPase R. Slayman C.W. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar). the are for the phosphorylation there is a the of mutations and a in the of Asp-378. to the J. J. Biol. PubMed Scopus Google by at a to the and could the of the of the ATPase, preventing of the C-terminal transmembrane into the lipid it has been shown that a role in the of proteins Smith J. Chem. PubMed Scopus Google and that mutations in have a to the of folding R. J. J. Biol. 1988; PubMed Scopus Google Scholar, J. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar). Based on the of protein it that the ATPases in a intermediate and as for CFTR A. N. J.R. S. J. PubMed Scopus Google with the mutations that or folding to this the of mutant protein that normal can the ER, the malfolded are be that the mutant are to on well ATPases such as S377A, and the it is whether the mutants of the plasma membrane are malfolded that the ER quality control or whether of the mutant proteins to of a of that secretory vesicles to the biogenesis mutants such as are of correct and for this biosynthetic it is to on the by which dominant lethal ATPase mutants cause co-expressed wild-type ATPase to be in the ER S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and F. FEBS Lett. 1997; PubMed Scopus Google this is that the mutant protein Helenius A. J. Cell Biol. 1992; PubMed Scopus Google to a of ER first this with the of ER that takes place of the ATPase A. A. J. Biol. Chem. 1993; Full Text PDF PubMed Google from C. R. J. Biol. Chem. 1992; Full Text PDF PubMed Google and the ER proteins R. M. L. J. J. Cell Biol. 1988; and J. J. L. J. Cell Sci. 1993; Google Scholar). by A. 1996; PubMed Scopus Google the of the ER can on the in is thought to as the to increased of these proteins in the expression of the dominant lethal PMA1 mutants the of the ER there to be a block in ER that the retention of the wild-type ATPase. shown in a recent in which secretory from cells the ATPase, a normal of proteins S. A. R. Slayman C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google that the biosynthetic block is to the ATPase or a of secretory have been for the of the block S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. 1996; PubMed Scopus Google Scholar). the wild-type ATPase with malfolded mutant ATPase, in the retention of the wild-type ATPase arrested to the abnormal of co-expressed mutant ATPase with of the ER biosynthetic such as a channel or a is to examine the of phosphorylation region mutants in the of these In as described there a the of mutant ATPase in the ER and the of the dominant lethal phenotype. an to this in the ER, and an on cell growth when with is that this mutant is well to the ER quality control with the of the wild-type ATPase at the plasma by this to the and of the region surrounding Asp-378 for biogenesis and folding of the ATPase. also to the quality control mechanisms that the of the ATPase from the ER, and that of those mechanisms be The recent of several ATPase biogenesis and S. M. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus (50) Google Scholar, A. Fink G.R. J. Cell Biol. 1995; PubMed Scopus Google Scholar, W. A. J. Cell Biol. 1997; PubMed Scopus Google serve as a of for such for the and for on yeast for the and expression for with and and for the of their and for this are also to and for the and