Red1p, a MEK1-dependent Phosphoprotein That Physically Interacts with Hop1p during Meiosis in Yeast

Abstract

The synaptonemal complex (SC) is a proteinaceous structure formed between pairs of homologous chromosomes during prophase I of meiosis. The proper assembly of axial elements (AEs), lateral components of the SC, during meiosis in the yeast,Saccharomyces cerevisiae, is essential for wild-type levels of recombination and for the accurate segregation of chromosomes at the first meiotic division. Genetic experiments have indicated that the stoichiometry between two meiosis-specific components of AEs inS. cerevisiae, HOP1 and RED1, is critical for proper assembly and function of the SC. A third meiosis-specific gene, MEK1, which encodes a putative serine/threonine protein kinase, is also important for proper AE function, suggesting that AE formation is regulated by phosphorylation. In this paper, we demonstrate that Mek1p is a functional kinasein vitro and that catalytic activity is an essential part of the meiotic function of Mek1 in vivo. Immunoblot analysis revealed that Red1p is aMEK1-dependent phosphoprotein. Co-immunoprecipitation experiments demonstrated that the interaction between Hop1p and Red1p is enhanced by the presence ofMEK1. Thus, MEK1-dependent phosphorylation of Red1p facilitates the formation of Hop1p/Red1p hetero-oligomers, thereby enabling the formation of functional AEs. The synaptonemal complex (SC) is a proteinaceous structure formed between pairs of homologous chromosomes during prophase I of meiosis. The proper assembly of axial elements (AEs), lateral components of the SC, during meiosis in the yeast,Saccharomyces cerevisiae, is essential for wild-type levels of recombination and for the accurate segregation of chromosomes at the first meiotic division. Genetic experiments have indicated that the stoichiometry between two meiosis-specific components of AEs inS. cerevisiae, HOP1 and RED1, is critical for proper assembly and function of the SC. A third meiosis-specific gene, MEK1, which encodes a putative serine/threonine protein kinase, is also important for proper AE function, suggesting that AE formation is regulated by phosphorylation. In this paper, we demonstrate that Mek1p is a functional kinasein vitro and that catalytic activity is an essential part of the meiotic function of Mek1 in vivo. Immunoblot analysis revealed that Red1p is aMEK1-dependent phosphoprotein. Co-immunoprecipitation experiments demonstrated that the interaction between Hop1p and Red1p is enhanced by the presence ofMEK1. Thus, MEK1-dependent phosphorylation of Red1p facilitates the formation of Hop1p/Red1p hetero-oligomers, thereby enabling the formation of functional AEs. Sexual reproduction requires the formation of haploid gametes so that fertilization can reconstitute the diploid chromosome number of the organism. The chromosome number of the cell is divided precisely in half by meiosis, a highly conserved and specialized type of cell division. Specifically, the reduction in chromosome number is accomplished by the segregation of homologous chromosomes to opposite poles at the first meiotic division. Proper disjunction requires crossovers (typically manifested cytologically as chiasmata) that provide the physical connections between homologs necessary for proper orientation on the Meiosis I spindle (1Bascom-Slack C.A. Ross L.O. Dawson D.S. Adv. Genet. 1997; 35: 253-284Crossref PubMed Scopus (49) Google Scholar). Mutants that interfere with the formation of crossovers produce chromosomally imbalanced gametes that are either inviable themselves or fuse to create inviable zygotes (2Roeder G.S. Genes Dev. 1997; 11: 2600-2621Crossref PubMed Scopus (683) Google Scholar). In most organisms, two requirements must be met for wild-type levels of crossovers to occur during meiosis. First, there must be recombination machinery to physically exchange the DNA from non-sister chromatids. Second, a tripartite proteinaceous structure called the synaptonemal complex (SC) 1The abbreviations used are: SC, synaptonemal complex; AE, axial element; HA, hemagglutinin; GST, glutathioneS-transferase; PCR, polymerase chain reaction; kb, kilobase(s); PMSF, phenylmethylsulfonyl fluoride. 1The abbreviations used are: SC, synaptonemal complex; AE, axial element; HA, hemagglutinin; GST, glutathioneS-transferase; PCR, polymerase chain reaction; kb, kilobase(s); PMSF, phenylmethylsulfonyl fluoride. must form between homologous chromosomes (3Von Wettstein D. Rasmussen S.W. Holm P.B. Annu. Rev. Genet. 1984; 18: 331-413Crossref PubMed Scopus (460) Google Scholar, 4Heyting C. Curr. Opin. Cell Biol. 1996; 8: 389-396Crossref PubMed Scopus (165) Google Scholar). SC formation is initiated by the condensation of sister chromatids along a protein core called an axial element (AE). Synapsis occurs when AEs are held together by the introduction of central region components to make mature SCs. In Saccharomyces cerevisiae, deletion of RED1, a gene encoding a component of AEs, eliminates AE formation and thereforered1 mutant phenotypes may indicate functions for AEs (5Rockmill B. Roeder G.S. Genetics. 1990; 126: 563-574PubMed Google Scholar). In red1 diploids, spore viability is drastically reduced, despite only a 4-fold reduction in crossing over, suggesting a role for AEs in packaging crossovers in such a way that the chiasmata can be used for directing Meiosis I chromosome segregation (5Rockmill B. Roeder G.S. Genetics. 1990; 126: 563-574PubMed Google Scholar, 6Mao-Draayer Y. Galbraith A.M. Pittman D.L. Cool M. Malone R.E. Genetics. 1996; 144: 71-86PubMed Google Scholar).RED1 also plays a role in a checkpoint that monitors whether recombination is progressing correctly or not. For example, mutations in the meiosis-specific recA homolog DMC1 cause arrest prior to the first meiotic division, presumably as a result of the formation of aberrant recombination intermediates (7Bishop D.K. Park D. Xu L. Kleckner N. Cell. 1992; 69: 439-456Abstract Full Text PDF PubMed Scopus (964) Google Scholar). In the absence ofRED1, this arrest is abolished despite the fact that the aberrant recombination intermediates are still being generated. It appears, therefore, that the packaging of recombination intermediates into some sort of RED1-dependent chromosomal structure is necessary for the recombination-monitoring checkpoint to work (8Xu L. Weiner B.M. Kleckner N. Genes Dev. 1997; 11: 106-118Crossref PubMed Scopus (188) Google Scholar). Understanding AE assembly is clearly a key to understanding axial element function. Genetic and cytological experiments have identified two other meiosis-specific genes, HOP1 and MEK1(also known as MRE4), that are important for formation of functional AEs in yeast. Evidence that the stoichiometry of Red1p to Hop1p is important in axial element formation comes from genetic experiments in which overexpression of RED1 in the presence of limiting amounts of Hop1p created a dominant negative phenotype (9Friedman D.B. Hollingsworth N.M. Byers B. Genetics. 1994; 136: 449-464PubMed Google Scholar). This dominant negative phenotype can also be produced by overexpressingRED1 in the presence of wild-type levels of HOP1but with limiting amounts of functional Mek1p (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar). Mek1p therefore appears to influence the Hop1p:Red1p stoichiometry. This paper presents biochemical data supporting the model that the interaction of Hop1p and Red1p with each other is promoted by phosphorylation of Red1p by Mek1p (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar). The genotypes of the strains used in this study are shown in Table I. All the strains are isogenic derivatives of the SK1-related diploid NH144 (11Hollingsworth N.M. Ponte L. Halsey C. Genes Dev. 1995; 9: 1728-1739Crossref PubMed Scopus (331) Google Scholar). Diploid strains mutated for RED1 or MEK1were created by first transforming S2683 and RKY1145, the two haploid parents of NH144, with the appropriate DNA fragments (12Rothstein R. Methods Enzymol. 1991; 194: 281-301Crossref PubMed Scopus (1093) Google Scholar) followed by mating. For the red1::LEU2 mutation, a 4.9-kbBglII/PstI fragment was purified from pNH119 (13Hollingsworth N.M. Johnson A.D. Genetics. 1993; 133: 785-797Crossref PubMed Google Scholar) and used for transformation. This insertion allele exhibits a null phenotype with respect to spore viability (other phenotypes have not been tested). A 3.6-kb XbaI/BamHI fragment from pTS1 carries the mek1Δ::LEU2 allele, and a 2.9-kb BamHI fragment from pTS21 was used to introducemek1Δ::URA3. These deletions remove 75 base pairs upstream of the ATG as well as approximately 75% of theMEK1 coding sequence. The presence of thered1::LEU2,mek1Δ::LEU2, andmek1Δ::URA3 alleles was confirmed both genetically and by Southern blot analysis. The ade2-Bglmutation was introduced by two-step gene replacement (12Rothstein R. Methods Enzymol. 1991; 194: 281-301Crossref PubMed Scopus (1093) Google Scholar) using pR943 (generously supplied by B. Rockmill, Yale University) cut withSpeI. Haploids containing the ade2-Bgl mutation and appropriate other markers were obtained by crosses. These haploids were then mated to give homozygous diploids. The mek1–974mutation was introduced into two differentmek1Δ::LEU2 haploids by two-step gene replacement using pNH235 digested with BssHI to target integration to the mek1Δ::LEU2 locus. Leu− 5FoaR colonies were then mated to create the mek1–974 diploid NH211. The red1::LEU2 mek1Δ::LEU2 homozygous diploid NH177 was constructed by crossing a MATa mek1Δ::LEU2 haploid with MATα red1::LEU2 haploid and dissecting tetrads from the resulting heterozygous diploid. Leu+ spore colonies that exhibited a non-parental di-type pattern for LEU2 were then mated to produce the double homozygote. make homozygous diploid and were with digested to target integration of the The resulting were mated to create the diploid. For and the and were digested with to target integration and into genetic were used Methods in A Scholar). have been Hollingsworth N.M. Johnson A.D. Cell. 1992; PubMed Scopus Google Scholar). and was using a as NH144 as as as NH144 as as NH144 as NH144 as NH144 as NH144 LEU2 in a for this study were by A N. Scholar) using the and are in Table deletion the polymerase chain was used to fragments or LEU2 with The fragments were digested and and to B. Roeder G.S. Genes Dev. 1991; PubMed Scopus Google Scholar) cut and to pTS21 and pTS1 N.M. Johnson A.D. Genetics. 1993; 133: 785-797Crossref PubMed Google N.M. Johnson A.D. Genetics. 1993; 133: 785-797Crossref PubMed Google N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google HOP1 N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google B. Roeder G.S. Genes Dev. 1991; PubMed Scopus Google with LEU2 LEU2 LEU2 B. Roeder G.S. Genes Dev. 1991; PubMed Scopus Google Genetics. PubMed Google in of in A.M. N. Genetics. 1997; PubMed Google in a fuse the gene to D.B. Johnson PubMed Scopus Google was to a fragment containing the The resulting fragment was digested with and into of to create The mutation was introduced into gene by M. Annu. Rev. Genet. PubMed Scopus Google Scholar). The 2.9-kb fragment from containing was to The presence of the mutation an that was used for with the The containing the allele was the and alleles of the the was first into the A fragment at to the ATG and at was by The was to an at the ATG of a The fragment was digested and and to make The 2.9-kb fragments containing or were then into cut with and to make and The alleles were to that mutations were introduced by either the or was using the followed by on an model for of the both the alleles were of theMEK1 the a fragment from (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar) was first to of with and the was with the fragment from either or resulting in the of approximately of the coding sequence. The resulting and were then digested with and and the fragments were into to make and The mek1–974 mutation was a by PubMed Scopus Google Scholar). The was cut and and into diploid (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar). The resulting was used in with at along the make a fragment from was into the digested with The first in the of the allele was the introduction of an in of by A fragment containing the of RED1 was digested and and (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar) A fragment from the RED1 gene with of upstream was into A.M. N. Genetics. 1997; PubMed Google Scholar). This the RED1 gene at the with of the in The allele was either to a or to a by the fragment from into and The is in the that was from were by D. of the protein were as The protein was in and purified to D.B. Hollingsworth N.M. Byers B. Cell. Biol. 18: PubMed Google Scholar). of purified protein was into two followed by a of by The resulting were to with a of the of when the gene D.B. Hollingsworth N.M. Byers B. Cell. Biol. 18: PubMed Google Scholar) were in This was when the were on The was also not using the from that are for Hop1p is that the protein is only in is were with colonies of the appropriate diploid and to at with containing were with colonies on was prior to to and of the were to The were into and for approximately with at an the were with and in to a of The were at for and then by for at in a to a was to the and the was by the levels of in of were with and the cell were at were as A.M. I. 1994; PubMed Scopus Google Scholar). were with and in of of was necessary to the as the were the not be The for this is not is not of of the were the be at and with in the to by analysis. PMSF, and were the and of was and the were for with on The was to a and were to of and The was for at and by at for The was divided into two to which either of or was followed by for at the of protein with were The were then for an at The were with of and in of protein The were at for and in a the were to and for Hop1p and using and The presence of the was using the from The of Hop1p and in the was as in the for the A of from the used in the was on the and with the appropriate the the were using a A of protein was for Hop1p and using the The of Hop1p and in the was then using the the of the in and the was For each of were used for of The were with protein as R. D. Cell Biol. 1997; PubMed Scopus Google Scholar). Genetic between and RED1 in the to the that phosphorylation of either Hop1p or Red1p by Mek1p facilitates the formation of Hop1p/Red1p hetero-oligomers, thereby the proper between the in the assembly of axial elements during meiosis (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar). this model was to the in which and with R. L. Kleckner Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). that the genetic of HOP1 overexpression in the presence of the mutant occur in as well as was necessary to the experiments in mutation was by using the diploid (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google and the gene was A mutation of to at was This mutation a to at which is the in the first conserved B.M. Methods in Scholar). The mek1–974 allele was introduced into the by two-step gene thereby the homozygous NH211. This diploid was with the used in the analysis (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google the were and tetrads were to spore in the the mek1–974 mutant is functional in with for the deletion 1992; PubMed Scopus Google and The spore viability is by on a The presence of HOP1 the mutant resulting in In overexpression of RED1 the number of from to the and the RED1 of spore phenotype are by analysis The between and was the result obtained when both HOP1 and RED1 were on the In the spore viability for In a phenotype to that for HOP1 was viability by HOP1 LEU2 LEU2 in a to Red1p Roeder G.S. Cell Biol. 1997; 136: PubMed Scopus Google Scholar) to the Red1p protein by analysis were an of the protein was The was in at the to of the The gene was then in two isogenic This insertion allele exhibits a null phenotype with to spore viability (13Hollingsworth N.M. Johnson A.D. Genetics. 1993; 133: 785-797Crossref PubMed Google Scholar). The haploids were mated to create diploid that is homozygous for allele gene on a number was into in a when and Hop1p are protein were from and protein of the appropriate for was with the in containing the These were meiosis, as by there being a wild-type of Hop1p in the was in This protein as this is from of NH144, an isogenic diploid homozygous for the RED1 gene, as well as from of in the allele is in are was to the of allele to the spore viability in produced is homozygous in was of the allele a of RED1 the was not as as overexpression of the allele of RED1 the allele was and a of RED1 function spore this was used in whether the of are of the protein was and for to protein the were in with or by and and the was using the In the was were protein was the were The of prior to the the of the that is the activity which for the of the and not a was that the phosphorylation is on the this is not the is demonstrated by the of and Roeder Roeder G.S. Genes Dev. PubMed Scopus Google Scholar) have shown that Red1p is a using the A for the which Red1p is whether the Mek1p is Mek1p activity was in The gene was to the of the and the allele was the of the a negative an allele of was created in which the for an in of the was mutated to This to mutation been shown to or activity in a number of other A.M. PubMed Scopus Google Scholar). The diploid was with or were for protein were and the and were purified using activity was by The of and during the was by the blot used for with A at the for was this is in activity was for the protein despite the fact that protein was for The reduction in phosphorylation of the protein that most of the activity is of Mek1p and not to a or to the were to the function of the The gene in the spore viability allele not also when into the that overexpression is not a for In were at the mek1Δ::LEU2 diploid Full of the spore viability was in the diploid with Table In the allele to mutation of the to at an allele of exhibits a null phenotype with to spore this mutation also Mek1p activity in we that the activity of Mek1p is essential for meiotic function. whether is a for the protein was in diploids. were from in the of in is as protein from was the there is a of the form of in both the of is in the diploid phosphorylation is therefore on This whether this is of phosphorylation of by Mek1p or whether an is the is a of is the spore viability of diploid is that is is this phosphorylation of is of the overexpression of the protein or whether occurs is not Co-immunoprecipitation experiments were to whether Hop1p and are physically in meiotic was for protein were and amounts of protein were with either or to and The for Hop1p was shown by the fact that is in from for is the is is when the is used for in the RED1 gene and or the is when from containing gene The Hop1p and Red1p were divided in by to and with either or to for of with Hop1p and In Hop1p with The is also when Hop1p was first with was in the complex whether phosphorylation of on the of to with the was using from there is Hop1p and in the absence of MEK1, as protein was used for the with theMEK1 diploid. the were with the protein amounts of Hop1p were from both strains when were with the a reduction in the of was with the In experiments the of with was a and reduction was in diploid with The reduction in the was not of a of in diploid as by the of in the from the diploid with and The of Hop1p with from a reduction of for the shown in and to in the diploid a with were and with and and the were The presence of therefore facilitates the interaction in vivo. Genetic in the to a model which that Mek1p phosphorylation facilitates the assembly of Red1p and in a stoichiometry necessary to functional AEs during SC formation (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar). this model was to the the and and HOP1 phenotypes in the with was that the model on the from the not to the For diploids, spore viability is to in 1992; PubMed Scopus Google Scholar) with in (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar). For spore viability is in both recombination is in Y. Galbraith A.M. Pittman D.L. Cool M. Malone R.E. Genetics. 1996; 144: 71-86PubMed Google Scholar, M. Genetics. 1993; 133: PubMed Google N.M. Byers B. Genetics. PubMed Google Scholar). the biochemical was therefore important to whether the and overexpression of HOP1 in in The mek1–974 allele used for the experiments in was by and introduced into allele is functional in the as in of HOP1 in the diploid in of the spore viability In overexpression of spore of HOP1 was shown to result in a phenotype to gene being with the that the two can each In the phenotype that of suggesting that this may be to the levels of a result with the recombination phenotype exhibited by in this The of the in indicated that be an appropriate in which to the stoichiometry model A of the stoichiometry model is that Mek1p is a serine/threonine protein This was to be by that Mek1p This activity is by of a conserved in the catalytic for mutation that function in vivo. the activity of Mek1p is an essential component of meiotic function. whether either or is aMEK1-dependent the two were by analysis for that may be of phosphorylation. to a form of Hop1p were In a form of that is to was The amounts of this form that may be the for Mek1p the presence of an be In the was only when the allele was suggesting that the presence of the may the of the The is to the functional form of the as is only when this is activity is in vivo. Genetic for Hop1p and Red1p as well as for Hop1p/Red1p (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar, N.M. Johnson A.D. Genetics. 1993; 133: 785-797Crossref PubMed Google Scholar, M. Genetics. 1993; 133: PubMed Google Scholar). The Hop1p been demonstrated using purified protein D.B. Hollingsworth N.M. Byers B. Cell. Biol. 18: PubMed Google Scholar). whether Hop1p and Red1p in meiotic experiments were The Hop1p both the and of The presence of in the Hop1p complex is not that Mek1p phosphorylation is not essential for the Hop1p/Red1p interaction of SC are in B. Roeder G.S. Genes Dev. 1991; PubMed Scopus Google Scholar). In the stoichiometry between Hop1p and Red1p is as there is with Hop1p Hop1p that with there is an in This is the result the of is this is the then when a of Hop1p is may not only the with which was also which was with the phosphoprotein. It is important to that experiments were with an allele of RED1 that is being and Roeder Roeder G.S. Cell Biol. 1997; 136: PubMed Scopus Google Scholar) have shown that overexpression of RED1 can result in a pattern of Red1p along the may be that the not the in strains containing levels of Mek1p phosphorylation of is not essential for the Hop1p/Red1p we have that facilitates the formation of the in the proper stoichiometry (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar). This was by the experiments in a diploid. is to amounts of Hop1p from meiotic from and diploids, the of was approximately 4-fold in the absence of was from and approximately Hop1p was in the The presence of clearly the Hop1p/Red1p formation is enhanced the for or an for Hop1p to be Co-immunoprecipitation of Hop1p and that the two physically in meiotic experiments not whether the are on chromosomes or not. that Hop1p and Red1p on AEs Roeder G.S. Cell Biol. 1997; 136: PubMed Scopus Google this The genetic data in (9Friedman D.B. Hollingsworth N.M. Byers B. Genetics. 1994; 136: 449-464PubMed Google Scholar) are with the of the that facilitates Hop1p/Red1p the be that the of with Hop1p and in the is an resulting from the levels of when is In the may be that the MEK1-dependent phosphorylation of Red1p some as function during meiosis. way to between two is to and the phosphorylation in Red1p and to such have for are The for AEs by may be conserved in for Mek1p and in and C. the of of the is a conserved of called the 1995; Full Text PDF PubMed Scopus Google Scholar). This is in and two protein of in the to DNA 1995; Full Text PDF PubMed Scopus Google Scholar). in there is an protein called that also an and B. It is to that is the functional homolog of Mek1p in that SC formation only occurs in The gene of a protein with to Hop1p mutant phenotypes and C. and in N.M. Byers B. Genetics. PubMed Google Scholar). may be the to Red1p in are meiosis-specific and are to in R.E. B. P.B. Cell 1994; PubMed Google Scholar, M. C. Cell. Biol. 1994; PubMed Scopus Google Scholar). the of both in the (10Hollingsworth N.M. Ponte L. Genetics. 1997; 147: 33-42Crossref PubMed Google Scholar, M. R.E. Park P.B. Biol. Cell. 1997; 8: PubMed Scopus Google Scholar). been shown to be that the pattern of phosphorylation is meiotic prophase M. C. 1995; PubMed Scopus Google Scholar). It is that the phosphorylation for the role in the protein stoichiometry in AEs that plays in the Hop1p/Red1p of Hop1p and Mek1p with Mek1 D. Mek1 Mek1 C. C. only a fragment is C. C. the fragment is Saccharomyces For C. only a fragment is For C. the fragment is in a Saccharomyces and for and on the are also to and for the protein in Table and Ponte and for