Fig. 4: Competition between Get4/5 and SRP with respect to ribosome-binding.
From: Ribosome-bound Get4/5 facilitates the capture of tail-anchored proteins by Sgt2 in yeast
a Schematic representation of in vitro analyzed RNCs. The C-terminus of the tail-anchor (TA) protein Sec22 was extended by 10–60 residues. Estimate of the nascent chain covered by the ribosomal tunnel43 (beige), Sec22 (dark gray, tail-anchor in red, C-terminal extension in light gray and white; for details see Supplementary Fig. 4a). b SRP binding to RNCs-Dap2-60 is enhanced in the absence of Get4/5. FLAG-tag pull-down reactions (see Methods and Supplementary Fig. 4b) with FLAG-tagged (+FLAG) or non-tagged (−FLAG) Dap2-60 or Dap2α−60 (control without TM domain18) were generated in translation extracts as indicated (Supplementary Fig. 4f). The relative amount of RNCs (Rpl4) and bound SRP (Srp54) was assessed by immunoblotting. c–d SRP reduces binding of Get4/5 to RNCs when the TA sequence is exposed outside of the ribosomal tunnel. RNCs carrying FLAG-tagged Sec22 variants (a) were generated in a wild type (c) or Δsrp54 translation extract (d). FLAG-pull-down reactions were performed as in b and intensities of immunostained bands were determined densitometrically. Relative ratios of Get4/Rpl31, Get5/Rpl31, and Srp54/Rpl31 were calculated and the ratio obtained for RNCs-Sec22 was set to 1; as the intensity of Srp54 on RNCs-Sec22 was below the detection limit (b.d.l.), RNCs-Sec22+10 were used for Srp54 normalization. See also Supplementary Fig. 4c. e Recruitment of Sgt2 to RNCs-Sec22 depends on Get4/5. FLAG-tag pull-down reactions were performed as in b. As indicated (+), purified His6Get4/Get5 (2 µM) was added prior to the translation reaction. f Sgt2 recruitment to RNCs-Sec22+60 is enhanced at high ribosome occupancy with Get4/5, or when SRP is absent. FLAG-tag pull-down reactions were performed and analyzed as in b with His6Get4/Get5 addition as in e. RNCs (Rps9 or Rpl31). g Quantification of Sgt2 recruitment to RNCs-Sec22+60. Ribosome-binding of Sgt2 was analyzed as in f. Shown are the normalized mean of five (wild type and Δsrp54) and two (wild type and Δsrp54 + His6Get4/Get5) biological replicates (bars) and the result of each experiment (dots) (see Methods).
