[View Larger Version of this Image]

Figure S1. Efficiency of the transport assay: effect of IMV formation method and transport buffer on pre-SufI transport efficiency. Features of our transport assay that may explain the high transport efficiencies obtained are: (1) the use of pre-SufI as a precursor substrate; (2) the formation of spheroplasts before French press treatment; (3) the use of low pressure for IMV formation, and a single pass through the French press cell; and (4) the use of organic buffers for the translocation reactions. The natural substrate pre-SufI was clearly transported more efficiently than the commonly used spTorA-GFP chimera (compare Fig. 1 G and with Fig. 2 A). The use of low pressure for IMV formation was essential (A). Due to the low pressure used, IMV yield was low unless spheroplasts were used (A). Phosphate buffer did not support in vitro translocation reactions with NADH as the energy source (B). (A) Effect of IMV formation method on pre-SufI transport efficiency. The highest pre-SufI transport efficiencies were observed when IMVs were made from spheroplasts at low pressure (lanes 9 and 10). Lower transport efficiencies were observed when IMVs were made from intact cells (lanes 5 and 6) or from spheroplasts at high pressure (lanes 7 and 8). (B) Effect of buffer on pre-SufI transport efficiency. Phosphate buffer did not support transport. All buffers were used at 50 mM, pH 7.0.