![A subset of the ER is degraded during meiosis and resynthesized in spores.(A) Quantification of cortically retained ER in experiments in Fig. 4, B–E. P values determined by χ2 test comparing cells expressing the PIL1-antiGFP tethering construct to those that do not. At least 30 cells were counted per strain background. ns, P > 0.05; ****, P < 0.0001. (B) Hierarchical clustering of z-score quantification of published protein measurements (Cheng et al., 2018) for all quantified proteins annotated for ER localization. Each row represents one protein, and each column is a time point in meiosis. Protein levels are at left and translation levels at right. Degradation of a subset of almost every ER-localized protein can be seen during mid- to late meiosis in multiple waves, occurring concomitant with or after ER collapse. Of the 658 proteins characterized for ER localization, we also observed robust synthesis (>50 RPKM [reads per kilobase of transcript per million mapped reads]) for 81.3% of them, with this synthesis occurring late in meiosis (after ER collapse) for 85.2% of this set. (C–E) To independently assess the turnover of an abundant ER protein during meiosis, we used an assay that takes advantage of the diploid status of meiotic cells (Eisenberg et al., 2018) by imaging cells with heterozygous tags marking the ER (RTN1-GFP/RTN1-WT) and histones (HTB1-mCherry/HTB1-WT). In this system, preexisting ER is marked by Rtn1-GFP, whereas newly synthesized ER following spore closure will be either marked or unmarked, depending on if the spore inherited RTN1-GFP or RTN1-WT, respectively. Consistent with the data in B, two spores per tetrad retained high levels of GFP signal, while GFP levels in the other two spores progressively declined, as opposed to the persistence of mCherry signal (and thus histone presence) in all four spores. (C) Time-lapse microscopy of cells heterozygous for RTN1-GFP and HTB1-mCherry imaged every 30 min in meiosis. Time 0 is defined as the time of spore individualization. Scale bar = 2 µm. (D) Quantification of the average GFP (left) and mCherry (right) signal for each spore for the cell in C. The GFP signal decrease in two spores indicates Rtn1 degradation. (E) Quantification of the average GFP signal for n = 10 cells at anaphase (ana) I, ana II, and the last imaged time point separated based on signal brightness (bright spore and dim spore). P values calculated by Student’s t test. *, P < 0.05; ****, P < 0.0001. (F–H) Western blot with samples taken from cells expressing the indicated GFP-tagged ER protein, taken at the indicated times following transfer to SPO and probed for GFP and hexokinase. Source data are available for this figure: SourceData FS4.](https://cdn.rupress.org/rup/content_public/journal/jcb/220/12/10.1083_jcb.202108105/6/jcb_202108105_figs4.png?Expires=1771349412&Signature=2e2bxQsGm9hWrQywiNhZY8D76iA3Rjbck5JQSCPpqLLM-pYvtBHYJS4~jfcVGH0EUCJzpyGbj6ExV-Fd2KQPPo-1i82O1~zJ54Mx25lEZKHExFnyryOS2qsmtr2kIdH9prwhfypOBvMFHeHyPnEH~YR7AaHH7w6JySZP9AQoqCqJ1LKUOrMUqT7eIuBZKLTZ5qW4YmAN3B5mYkof~T-CxKQcgiZS4pamich7Qmg97NNWeHFs2oGJVEMwaiSXCSCV8dpw5tw2S-GVzWrgo9GQt5VELCMXkJxRBj5wQ-Hi8sM26Q6JGWUA8DHtbzMwzS8mj7T1Z7vnT5WpzaI~cLcRKQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
A subset of the ER is degraded during meiosis and resynthesized in spores. (A) Quantification of cortically retained ER in experiments in Fig. 4, B–E. P values determined by χ2 test comparing cells expressing the PIL1-antiGFP tethering construct to those that do not. At least 30 cells were counted per strain background. ns, P > 0.05; ****, P < 0.0001. (B) Hierarchical clustering of z-score quantification of published protein measurements (Cheng et al., 2018) for all quantified proteins annotated for ER localization. Each row represents one protein, and each column is a time point in meiosis. Protein levels are at left and translation levels at right. Degradation of a subset of almost every ER-localized protein can be seen during mid- to late meiosis in multiple waves, occurring concomitant with or after ER collapse. Of the 658 proteins characterized for ER localization, we also observed robust synthesis (>50 RPKM [reads per kilobase of transcript per million mapped reads]) for 81.3% of them, with this synthesis occurring late in meiosis (after ER collapse) for 85.2% of this set. (C–E) To independently assess the turnover of an abundant ER protein during meiosis, we used an assay that takes advantage of the diploid status of meiotic cells (Eisenberg et al., 2018) by imaging cells with heterozygous tags marking the ER (RTN1-GFP/RTN1-WT) and histones (HTB1-mCherry/HTB1-WT). In this system, preexisting ER is marked by Rtn1-GFP, whereas newly synthesized ER following spore closure will be either marked or unmarked, depending on if the spore inherited RTN1-GFP or RTN1-WT, respectively. Consistent with the data in B, two spores per tetrad retained high levels of GFP signal, while GFP levels in the other two spores progressively declined, as opposed to the persistence of mCherry signal (and thus histone presence) in all four spores. (C) Time-lapse microscopy of cells heterozygous for RTN1-GFP and HTB1-mCherry imaged every 30 min in meiosis. Time 0 is defined as the time of spore individualization. Scale bar = 2 µm. (D) Quantification of the average GFP (left) and mCherry (right) signal for each spore for the cell in C. The GFP signal decrease in two spores indicates Rtn1 degradation. (E) Quantification of the average GFP signal for n = 10 cells at anaphase (ana) I, ana II, and the last imaged time point separated based on signal brightness (bright spore and dim spore). P values calculated by Student’s t test. *, P < 0.05; ****, P < 0.0001. (F–H) Western blot with samples taken from cells expressing the indicated GFP-tagged ER protein, taken at the indicated times following transfer to SPO and probed for GFP and hexokinase. Source data are available for this figure: SourceData FS4.
