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. 2020 Jun 8;295(30):10406–10419. doi: 10.1074/jbc.RA120.012575

Figure 4.

Figure 4.

The transcription factor Rme1 is N-glycosylated in a signal peptide-independent manner. A, Western blot analysis for the screening of signal peptide- and transmembrane domain-less proteins that undergo N-glycosylation in the ste24Δ mutant. V5-tagged candidate proteins were expressed and analyzed as described above. B, Endo H treatment of Rme1. Rme1-V5 was expressed in WT and ste24Δ cells. Cell extracts were mock treated (−) or digested (+) with Endo H and resolved by SDS-PAGE. Rme1 was detected as described above. The bar graph shows the relative ratio of glycosylated versus total Rme1 proteins. n represents the number of biological replicates. C, Western blot analysis of Rme1 expressed in the spc2Δ mutant. Rme1-V5 was expressed in the indicated cells. Extracts from these cells were analyzed as described above. The bar graph shows the relative ratio of glycosylated versus total Rme1 proteins. n represents the number of biological replicates. D, Western blot analysis of Rme1 expressed in the sec61-41 mutant. Rme1-V5 was expressed in the indicated cells at the permissive temperature (30 °C). Extracts from these cells were analyzed as described above. E, Western blot analysis of Rme1 expressed in the sec66 and sec72 mutants. Rme1-V5 was expressed in the sec66 and sec72 mutants. Extracts from these cells were analyzed as described above. F, Western blot analysis of the N-terminally truncated mutant of Rme1. V5-tagged Rme1, Rme1ΔN20, Rme1ΔN40, Rme1ΔN60, and Rme1ΔN80 were expressed in ste24Δ cells. Extracts from these cells were analyzed as described above. G, Protease protection assay of Rme1. Rme1-V5 and Pdi1-HA were coexpressed in WT and ste24Δ cells. The protease protection assay was performed using these strains. Samples were resolved by SDS-PAGE, and Rme1-V5 or Pdi1-HA was visualized by immunoblotting using an anti-V5 or an anti-HA antibody, respectively. The bar graph shows the relative abundance of trypsin-treated Rme1 normalized by Pdi1-HA. H, Western blot analysis of Rme1 expressed in the ste24Δ png1Δ mutant. Rme1-V5 was expressed in WT, png1Δ, ste24Δ, and ste24Δ png1Δ cells. Extracts from these cells were analyzed as described above. The bar graph shows the amount of Rme1 relative to that of Pgk1 expressed in ste24Δ and ste24Δ png1Δ cells. n represents the number of biological replicates.