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. 2003 Oct;23(20):7403–7414. doi: 10.1128/MCB.23.20.7403-7414.2003

FIG. 4.

FIG. 4.

NatA contacts a variety of ribosome-bound nascent chains. (A) Cross-link reactions were performed with low-salt-treated RNCs carrying the prepro α-factor, Rpl4A, or Rpl4A-S(2)K as a nascent chain. Aliquots of the cross-link reactions were subjected to immunoprecipitation under denaturing conditions. Anti-ssb1 (α-ssb1), anti-nat1 (α-nat1), or preimmune serum (pre) was used for the immunoprecipitations. The material bound to the respective antibody was run on 10% Tris-Tricine gels and subsequently analyzed by autoradiography. (B) RNCs carrying Rpl4A-S(2)K were generated and subsequently separated from soluble proteins by centrifugation through a sucrose cushion under low-salt conditions (LS-Sup and LS-ribos), under high-salt conditions (HS-Sup and HS-ribos), and after treatment with puromycin (HP-Sup and HP-ribos) as described in Materials and Methods. Each sample was subjected to a cross-link reaction and subsequently to immunoprecipitation under denaturating conditions with anti-nat1. The material bound to anti-nat1 (upper panel) and a total of the cross-linking reaction (lower panel) was run on a 10% Tris-Tricine gel and subsequently analyzed by autoradiography. Note, as shown in lane 1, a fraction of Rpl4A-S(2)K was not associated with the ribosome under low-salt conditions. This fraction does not contain NatA, which is retained in the LS-ribos fraction (compare Fig. 2B). (C) Low-salt RNCs carrying different length yeast malate dehydrogenase (mdh1), prepro α-R(2)K, or Rpl4A-S(2)K as indicated were subjected to cross-linking reactions. The numbers indicate the length of the nascent chains in amino acids (aa). Each sample was split into three aliquots and subjected to immunoprecipitation reactions under denaturating conditions with anti-nat1, anti-ssb1, or anti-beta-nac antibodies. The material bound to the respective antibody was run on 10% Tris-Tricine gels and subsequently analyzed by autoradiography. Nat1p-CL, cross-link of the nascent chain to Nat1p; Ssb1/2p-CL, cross-link of the nascent chain to Ssb1/2p; beta-NAC-CL, cross-link of the nascent chain to beta-NAC. (D) Protein sequences of the nascent chains used for cross-linking experiments.