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. Author manuscript; available in PMC: 2022 Dec 20.
Published in final edited form as: Dev Cell. 2021 Nov 22;56(24):3380–3392.e9. doi: 10.1016/j.devcel.2021.10.023

Figure 5. HAP2 expression and localization depend on MAR1, but MAR1 properties are independent of HAP2.

Figure 5.

(A-B) MAR1 expression and localization are independent of HAP2. (A) Immunoblots of MAR1-FLAG (upper panel) in lysates of mar1;MAR1-FLAG;hap2;HAP2-HA(−) gametes (left side) and mar1;MAR1-FLAG;hap2(−) gametes (right side). Tubulin is shown as a loading control (lower panel). (B) MAR1-FLAG immunolocalization in mar1;MAR1-FLAG;hap2;HAP2-HA(−) gametes (left), and mar1;hap2;MAR1-FLAG(−) gametes (middle) by fluorescence microscopy. Negative control (right) was WT(−) gametes expressing neither HAP2-HA nor MAR1-FLAG. (C-F) HAP2 expression and mating structure localization are impaired in minus gametes lacking MAR1. (C) Representative immunoblots from lysates of HAP2-HA-expressing (left panel) or HAP2-FLAG-expressing (right panel) minus gametes in the absence (left lanes) or presence (right lanes) of WT MAR1. Controls (right lanes) were hap2::HAP2-HA(−) and hap2::HAP2-FLAG(−) gametes which contained the endogenous WT MAR1 gene; lower panel shows tubulin (tub) loading controls. (D) Quantification of total HAP2-HA and HAP2-FLAG in immunoblots that was normalized to tubulin for each sample. Median HAP2 expression (bar) in mar1(−) gametes was 49% of that in minus gametes expressing WT MAR1; Wilcoxon Signed Rank test, *P=0.04. Each open circle represents a biological replicate mar1(−) sample (n=8) and is expressed as a percentage of the median signal present in WT MAR1(−) samples (n=5). (E) Quantification of the percentage of total HAP2 present in the upper isoform of the HAP2 doublet that was determined from immunoblot signals of HAP2-HA or HAP2-FLAG expressed in mar1(−) or WT MAR1(−) gametes. The mean percent of cell surface HAP2 in mar1(−) gametes was 36% (n=15) compared to 50% in WT MAR1(−) gametes (n=17); two-tailed unpaired t test with Welch’s correction; ****P<0.0001; bars are mean; error bars, SD. (F) Confocal z-stack composite images of anti-HA immunostained mar1;HAP2-HA(−) gametes (lacking MAR1, right), hap2::HAP2-HA(−) gametes (containing WT MAR1, middle), and control WT(+) gametes (left). The HA immunofluorescence of the mar1;HAP2-HA(−) gametes was substantially dimmer than that of the hap2::HAP2-HA(−) gametes, and the relative brightness was increased here to allow visualization of the altered HAP2-HA distribution (see also Fig. S5).