Limits...
Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis.

Voelkel-Meiman K, Johnston C, Thappeta Y, Subramanian VV, Hochwagen A, MacQueen AJ - PLoS Genet. (2015)

Bottom Line: While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery.This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast.Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut, United States of America.

ABSTRACT
Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

No MeSH data available.


Zip3-MYC and Zip4-HA levels exhibited by K. l. ZIP1-expressing cells resemble zip1  levels.Sporulating cultures of S. cerevisiae meiotic cells carrying one copy of ZIP3-MYC and ZIP4-HA and expressing S. c. ZIP1 (AM3362), a zip1  allele (AM3363) or K. l. ZIP1 (AM3361) were surface-spread on glass slides at 24 hours. AM3362 and AM3361 strains are homozygous for an ndt80  allele, and thus will not progress beyond the pachytene stage of meiotic prophase. AM3363 sporulating cultures are enriched for pachytene owing to the fact that zip1 meiotic cells trigger the meiotic prophase checkpoint. Immunolocalization was used to label the chromosomal axis protein Red1 (white, blue), Zip3-MYC (red) and Zip4-HA (green) on surface-spread meiotic chromosomes. See (S2 Fig) for quantification of Zip3-MYC and Zip4-HA foci and frequency of co-localization. Scale, 1 micron.
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pgen.1005335.g005: Zip3-MYC and Zip4-HA levels exhibited by K. l. ZIP1-expressing cells resemble zip1 levels.Sporulating cultures of S. cerevisiae meiotic cells carrying one copy of ZIP3-MYC and ZIP4-HA and expressing S. c. ZIP1 (AM3362), a zip1 allele (AM3363) or K. l. ZIP1 (AM3361) were surface-spread on glass slides at 24 hours. AM3362 and AM3361 strains are homozygous for an ndt80 allele, and thus will not progress beyond the pachytene stage of meiotic prophase. AM3363 sporulating cultures are enriched for pachytene owing to the fact that zip1 meiotic cells trigger the meiotic prophase checkpoint. Immunolocalization was used to label the chromosomal axis protein Red1 (white, blue), Zip3-MYC (red) and Zip4-HA (green) on surface-spread meiotic chromosomes. See (S2 Fig) for quantification of Zip3-MYC and Zip4-HA foci and frequency of co-localization. Scale, 1 micron.

Mentions: Further support for the conclusion that K. l. Zip1 fails to assemble SC in S. cerevisiae came from staining of the axial element protein, Red1, on surface-spread meiotic chromosomes. Red1 labels the axes of meiotic prophase chromosomes [61]; because the SC structure brings homolog axes into intimate alignment along their entire lengths, the closely apposed Red1-labeled axes of partner homologs in wild-type meiotic pachytene bivalents appear as a single linear structure along their full-lengths (Fig 5, top left). In contrast, meiotic pachytene chromosomes from zip1 cells exhibit loosely-associated chromosome axes labeled by Red1 (Fig 5, bottom left) [10]. The Red1-labeled “loops” apparent in such synapsis-defective mutants correspond to homolog axes joined in intimate alignment only at sporadic positions along the chromosomes (these “axial associations” are presumably where a crossover event has been established) [62]. The Red1-stained chromosome axis patterns exhibited by surface-spread meiotic chromosomes from K. l. ZIP1-expressing S. cerevisiae cells appeared indistinguishable from those seen in zip1 cells, consistent with an absence of mature SC structure (Fig 5, middle left).


Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis.

Voelkel-Meiman K, Johnston C, Thappeta Y, Subramanian VV, Hochwagen A, MacQueen AJ - PLoS Genet. (2015)

Zip3-MYC and Zip4-HA levels exhibited by K. l. ZIP1-expressing cells resemble zip1  levels.Sporulating cultures of S. cerevisiae meiotic cells carrying one copy of ZIP3-MYC and ZIP4-HA and expressing S. c. ZIP1 (AM3362), a zip1  allele (AM3363) or K. l. ZIP1 (AM3361) were surface-spread on glass slides at 24 hours. AM3362 and AM3361 strains are homozygous for an ndt80  allele, and thus will not progress beyond the pachytene stage of meiotic prophase. AM3363 sporulating cultures are enriched for pachytene owing to the fact that zip1 meiotic cells trigger the meiotic prophase checkpoint. Immunolocalization was used to label the chromosomal axis protein Red1 (white, blue), Zip3-MYC (red) and Zip4-HA (green) on surface-spread meiotic chromosomes. See (S2 Fig) for quantification of Zip3-MYC and Zip4-HA foci and frequency of co-localization. Scale, 1 micron.
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pgen.1005335.g005: Zip3-MYC and Zip4-HA levels exhibited by K. l. ZIP1-expressing cells resemble zip1 levels.Sporulating cultures of S. cerevisiae meiotic cells carrying one copy of ZIP3-MYC and ZIP4-HA and expressing S. c. ZIP1 (AM3362), a zip1 allele (AM3363) or K. l. ZIP1 (AM3361) were surface-spread on glass slides at 24 hours. AM3362 and AM3361 strains are homozygous for an ndt80 allele, and thus will not progress beyond the pachytene stage of meiotic prophase. AM3363 sporulating cultures are enriched for pachytene owing to the fact that zip1 meiotic cells trigger the meiotic prophase checkpoint. Immunolocalization was used to label the chromosomal axis protein Red1 (white, blue), Zip3-MYC (red) and Zip4-HA (green) on surface-spread meiotic chromosomes. See (S2 Fig) for quantification of Zip3-MYC and Zip4-HA foci and frequency of co-localization. Scale, 1 micron.
Mentions: Further support for the conclusion that K. l. Zip1 fails to assemble SC in S. cerevisiae came from staining of the axial element protein, Red1, on surface-spread meiotic chromosomes. Red1 labels the axes of meiotic prophase chromosomes [61]; because the SC structure brings homolog axes into intimate alignment along their entire lengths, the closely apposed Red1-labeled axes of partner homologs in wild-type meiotic pachytene bivalents appear as a single linear structure along their full-lengths (Fig 5, top left). In contrast, meiotic pachytene chromosomes from zip1 cells exhibit loosely-associated chromosome axes labeled by Red1 (Fig 5, bottom left) [10]. The Red1-labeled “loops” apparent in such synapsis-defective mutants correspond to homolog axes joined in intimate alignment only at sporadic positions along the chromosomes (these “axial associations” are presumably where a crossover event has been established) [62]. The Red1-stained chromosome axis patterns exhibited by surface-spread meiotic chromosomes from K. l. ZIP1-expressing S. cerevisiae cells appeared indistinguishable from those seen in zip1 cells, consistent with an absence of mature SC structure (Fig 5, middle left).

Bottom Line: While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery.This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast.Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut, United States of America.

ABSTRACT
Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data suggest that features of S. cerevisiae Zip1 or of the assembled SC in S. cerevisiae normally constrain MutLγ to preferentially promote resolution of MutSγ-associated recombination intermediates.

No MeSH data available.