Fig. 1: Hel2 is associated with translating ribosomes and collided disomes.

a Hel2 binds to translating ribosomes and collided disomes. Polysome profiles were generated by sucrose gradient sedimentation (see Methods) with untreated log-phase extract (wild type_log, orange), after RNase treatment (wild type_log/RNase, magenta), or with extract derived from glucose depleted cells (wild type_-glc, light green). The approach is detailed in Fig. S1a. b Upon overexpression, a large fraction of Hel2 is bound to translating ribosomes. Polysome profiles with log-phase extract from Hel2↑ cells without (Hel2↑_log, orange) or after RNase treatment (Hel2↑_log/RNase, magenta). For immunodetection of overexpressed Hel2, loading was reduced to 1/3 of the wild type. An A₂₆₀ trace of wild type_log/RNase (dotted gray line), recorded side by side, is shown as a control. c Hel2 does not bind to non-translating ribosomes. Cells were starved for glucose and subsequently sucrose density sedimentation was performed with untreated extract (wild type_-glc, light green) and after RNase treatment (wild type_-glc/RNase, dark green). a–c A₂₆₀ profiles, Hel2 immunoblots, Hel2 distribution profiles (see Methods), and immunoblots of the 60S ribosomal protein Rpl4 and the 40S ribosomal protein Rps9. The total (tot) corresponds to 5% of the extract loaded onto the gradient. The position of 40S, 60S, 80S, disomes, and higher order polysomes is indicated. Clubs indicate Hel2 bands, gray asterisks indicate a background band recognized by α-Hel2 (Fig. S1b). The disome region of A₂₆₀ traces is marked by a white spotlight.