Figure 8.

Import Efficiencies of Fixed-Diameter Probes.

(A) and (B) Eight-minute import reactions of fluorescein (F)-labeled and Undecagold (UG)-labeled RSSUFHC (A) and mSA-labeled RSSUHC-Bt (B) are shown. RSSUHC-Bt (8.3 μg/mL in import reaction) was incubated with a 70-fold molar excess of mSA in import buffer prior to import. All reactions were stopped after 8 min with cold 400 μg/mL thermolysin and incubated 1 h on ice. Samples were subsequently treated as in Figures 6 and 7. Mature RSSU (mRSSUHC-Bt) and mSA were quantified to illustrate their equimolar import ratio.

(C) Import efficiencies of all three labeled proteins (defined as the labeled:unlabeled mature protein ratio) were plotted as a function of probe size (black circles). The internal control unlabeled protein was used for fluorescein and Undecagold quantifications. Error bars (sd) represent multiple replicates from two independent experiments. For comparison, tp22DHFR/FMTX import efficiency was plotted as the ratio of tp22DHFR import in the presence and absence of FMTX derived from Figure 3H (green circle, error bar = sd, n = 3). Protein dimensions were plotted as minor axis diameters with error bars representing sd away from a perfectly circular cross section in the minor axis plane. Mitoplast TIM complex import rates for fluorescein (13 Å), Undecagold (20 Å), and Nanogold (26 Å) size probes were taken from Schwartz and Matouschek (1999). The Undecagold import efficiency was plotted as the labeled:unlabeled ratio of preprotein extent of import after 4 min since the authors noted that the initial import rates overestimate the import efficiency. Bacterial SecYEG transport rates for tetraarylmethane derivative size probes (TAMs) were replotted from Bonardi et al. (2011) with the unlabeled substrate transport rate normalized to 1. Internally cross-linked BPTI translocation efficiency through TOC/TIC, TIM, and SecYEG complexes (cyan circle/square/triangle) and DHFR/MTX translocation efficiency through TIM and SecYEG complexes (green square/triangle) also derive from previous publications (Eilers and Schatz, 1986; Schiebel et al., 1991; Jascur et al., 1992; Arkowitz et al., 1993; Clark and Theg, 1997). Asterisk indicates nonspecific chloroplast protein.