(A) Schematic of S. cerevisiae (Sc) chromosome axis proteins Hop1 and Red1, and Zygosaccharocmyces zouxii (Zr) Red1. Yellow regions indicate Hop1-binding closure motifs (West et al., 2018). For S. cerevisiae Red1, the positions of two previously-identified mutations in the C-terminal domain that disrupt axis function, I743A (Eichinger and Jentsch, 2010) and I758R (Lin et al., 2010), are shown. See Figure 1—figure supplement 1A for sequence alignment of the Red1 C-terminal domain. (B) SEC-MALS analysis of purified His6-MBP-Zr Red1705-798. Calculated molecular weight of a monomer = 55.9 kDa; Measured molecular weight = 4783 kDa (~85 mer). (C) Representative negative-stain electron micrographs of purified untagged Zr Red1705-798. See Figure 1—figure supplement 2A for additional full micrographs, and Figure 1—figure supplement 2B for micrographs of His6-MBP-Zr Red1705-798. (D) SEC-MALS analysis of purified His6-MBP-Zr Red1705-791 and His6-MBP-Zr Red1705-798 I715R. (E) SEC-MALS analysis of purified Zr Red1705-791 and Zr Red1705-798 I715R (as in panel D, but with His6-MBP tag removed). (F) Table summarizing SEC-MALS results from (D) and (E). (G) Schematic of Zr Red1 C-terminal domain oligomerization. Wild-type Zr Red1705-798 forms homotetramers that further oligomerize into extended filaments. Removal of the C-terminal seven amino acids (Zr Red1705-791) or mutations of I715 to arginine (Zr Red1705-798 I715R) results in loss of filament formation but maintenance of tetramer formation. (H) Representative surface-spread mid-meiotic prophase nuclei from wild-type (top row), and red1 mutant alleles: red1-Sc1-734:Zr707-798 (second row), red1-Sc1-734:Zr707-791 (third row), and red1-Sc1-734:Zr707-798 I715R] (bottom row). Spore viability for each homozygous strain is shown in blue (n = 52–128, see Materials and Methods). Mid-meiotic prophase chromosomes are stained with DAPI to label DNA (white), anti-Red1 (magenta), and anti-Gmc2 (green). Scale bar, 1 μm. See Figure 1—figure supplement 3B–E for additional images. (I) Quantification of the distribution of Red1 on meiotic chromosomes at 3 hr and 5 hr after introduction into sporulation medium (n = 30–50 cells for each strain and time-point). ‘foci + short linear’=cells with abundant foci and short linear stretches of Red1 staining; ‘abundant foci’=cells with more than 25 strong Red1 foci; ‘few foci’=cells with fewer than 25 weak Red1 foci. See Figure 1—figure supplement 3F–H for further quantification of Red1, Gmc2, and polycomplex assembly.