Fig. 3. The ability of Ltn1p to access lysines
outside the exit tunnel is primarily determined by the distance of the lysines from the
C terminus. (A and C) IB of TEV-treated
GFPLys-free stalling constructs with XTEN linkers
of the indicated length inserted after the lysines.
(B) IB of a stalling construct with a lysine
positioned between two unstructured sequences. DHFR was used to stabilize the N terminus
of the construct. (D) Quantification of protein
levels from the stalling constructs in (C) (left).
Shown is the percentage of stalling construct
remaining in wt or rqc2mut cells relative to rqc2D
cells as a function of the distance between the
lysines and the R12 stalling site (mean ± SD,
N = 3). Schematic of Ltn1p’s accessible region
(shaded area) (right). (E) IB of non-stop GFPLys-free
Fig. 4. CAT-tail–dependent degradation of endogenous RQC substrates. (A) Fraction of
stalling positions leading to an RQC-degradable nascent polypeptide in the presence (solid line) or
absence (dashed line) of CAT-tails, graphed as a function of the number of amino acids accessible
to Ltn1p and assuming a fixed exit tunnel length of 35 amino acids. The arrow shows the increase in
RQC-degradable substrates in the presence of CAT-tails at the estimated Ltn1p reach of 12 amino
acids. (B) Growth phenotypes of RQC mutant strains in the absence or presence of cycloheximide
(CHX). (C) Model for the function of CAT-tails in vivo.