(A) In substrates with otherwise all-glycine cassettes, fraction intracellular degradation depends on side-chain identity at tail-position 4. (B) Comparison of degradation in vivo for substrates with Thr or Val at tail-position four or Glu or Gln at tail-position 4 (Student’s two-tailed t-test significance; Val/Thr: t = 6.37, df = 4; Glu/Gln: t = 5.47, df = 4). (C) Vmax values from Michaelis-Menten analysis of degradation of purified substrates. (D) Effects of position-4 residues, color-coded by side-chain properties, on Vmax. (E) Comparison of degradation in vitro between substrates with Ala, Ser, Cys, Thr, or Val at tail-position four or Glu or Gln at tail-position 4 (Student’s two-tailed t-test significance; Val/Thr: t = 13.3, df = 4; Glu/Gln: t = 5.49, df = 4). (F) ATP cost of degrading substrates with Ala, Cys, Thr, Val, Glu, or Gln at tail-position 4. With the exception of panel A, where Gly12 and GA values represent averages (± S.D.) of nine biological replicates, all values represent three biological replicates.