Amino-terminal nascent chain ubiqutination is important for NCA formation. (A) Ubiquitinated proteins were affinity purified by nickel beads from cells coexpressing a stalled nascent Rpt1 chain (RNC) (RNC construct above blot) and His6-tagged ubiquitin from the CUP1 promoter. The input extract (TE) or Ubi-affinity proteins (Ubi) were detected by western blotting with antibodies to the amino-terminal Flag-tag. The discrete Rpt1-RNC is ubiquitinated (Ubi-Rpt1-RNC). Polyubiquitinated forms of the Rpt1-RNC are additionally visible (Poly Ubi-Rpt1-RNC). (B) Expression of the Rpt1-RNC without (1) and with (2) the first six lysines mutated to arginine (mutations shown above the blot) (Ubiless-Rpt1-RNC) monitored by western blotting with anti-Flag antibodies. (C) Polysome profiling of cells expressing the Rpt1-RNC or the Ubiless-Rpt1-RNC (as in B). Fractions were visualized with anti-Flag antibodies. (TE) Total extract, (F) free fractions, (M) 80S monosomes, (P1) light polysomes, (P2) heavy polysomes (the profile for the Ubi-Rpt1-RNC is shown above the blots with an indication of the analyzed fractions).
