Figure 1.
Cell-free translation and purification of AAC-containing proteoliposomes.
A) Schematic illustration of the experimental process. [35S]AAC is translated in a wheat germ-based system in the presence of SUVs and the sample is subjected to SGU. Buoyant proteoliposomes remain at the top of the gradient, whereas aggregated protein and ribosomes from the translation reaction pellet. B) The extent of AAC integration depends on liposome concentration. [35S]AAC was translated in the presence of variable SUV concentrations as indicated and samples from each fraction were resolved by SDS-PAGE. Fractions 1–4 indicate fractions collected from top to bottom of the gradient. Normalized band intensities are shown below each lane of the gel.
Figure 2.
Assays for AAC integration into liposomes.
A) Proteoliposomes containing the indicated radiolabeled proteins were incubated in the presence and absence of 100 mM Na2CO3 (pH 11.5). Following carbonate extraction, pellets and TCA-precipitated supernatants were resolved by SDS-PAGE. The percentage of protein per sample has been indicated. B) Site-specific labeling of AAC monocysteine variants with thiol-reactive NBD. AAC constructs with single cysteine residues on either the second cytosolic loop (AAC L219C, top panel) or transmembrane segment four (AAC L194C, bottom panel) were translated in the presence or absence of liposomes as indicated and subjected to a labeling reaction time course with IANBD. After quenching the reaction, samples were resolved by SDS-PAGE and the relative extent of labeling was quantified by in-gel fluorometry. The mean labeling efficiencies of four independent measurements is shown with standard deviations. Where indicated, the extent of labeling between samples with and without liposomes was significantly different (p<0.01 [**] and p<0.001 [***]) based on two-tailed t-tests.
Figure 3.
In vitro translated AAC in proteoliposomes is transport competent.
Liposomes (A) or AAC-containing proteoliposomes (B, C) pre-loaded with ATP were incubated in the translation reaction to allow transport to occur. Vesicles were then purified by size-exclusion chromatography to remove unencapsulated nucleotide and assayed for total ATP content by luminescence immediately after disruption with detergent (0.2% triton X-100). A) Luminescence measurements of ATP-loaded liposomes pre-incubated with buffer only (white bar) or with detergent (hatched bar) registered a higher luminescence signal (p<0.0001 [****], two-tailed t-test) following disruption of the vesicles to release ATP. B) AAC-proteoliposomes with encapsulated ATP were prepared in the presence or absence of CAT as indicated and subjected to pretreatment with buffer only (white bars) or detergent (hatched bars) prior to luminescence measurements. The internal ATP content of proteoliposomes without CAT was significantly lower than samples prepared with CAT (p<0.01 [**], two-tailed t-test). The presence of CAT itself had no effect on the luminescence readings. C) AAC-proteoliposomes were prepared as in panel B, then incubated with buffer only (white bar) or 0.2 mM ADP (hatched bar), purified by a second round of gel filtration, and assayed for total luminal ATP content after detergent disruption. The internal ATP content of proteoliposomes subjected to ADP incubation was significantly lower than samples incubated with buffer only (p<0.01 [**], two-tailed t-test). All relative luminescence intensity measurements are means from a minimum of three independent experiments with standard deviations.
Figure 4.
The presence of CL in liposomes enhances AAC synthesis and membrane integration.
A) Liposome dependence of total protein synthesis. Cell-free translation reactions were programmed with mRNA encoding AAC or Tim9 in the absence of liposomes (“X”) or in the presence of liposomes at the final lipid concentration shown. B) Effect of CL concentration on AAC-liposome association. AAC translation reactions were conducted in the presence of liposomes containing variable mol% CL as indicated and purified by SGU. The relative amounts of co-isolated [35S]AAC are shown as mean values from a minimum of three independent experiments with standard deviations. C) Effect of CL concentration on AAC integration. [35S]AAC-containing proteoliposomes with variable amounts of CL were prepared and purified by SGU as in panel B. A subset of the samples were subjected to mock (buffer only) treatment (left panel) and the remaining samples were subjected to carbonate or urea extraction (right panel). Values shown are average band intensities normalized with respect to the highest value for liposome associated (left) or fully integrated (right) sample sets from three independent experiments. The relative amount of [35S]AAC in mock-treated samples (left), with 16 or 24 mol% CL, is significantly higher (p<0.05 [*], two tailed t-test) than with 0 mol% CL. In carbonate- and urea- treated samples (right) the difference in the relative amount of [35S]AAC present in the 16 and 24 mol% CL samples (compared to 0%) is even greater than in the untreated samples (p<0.01 [**] or p<0.001 [***], two-tailed t-test).
Figure 5.
Liposome-assisted integration of AAC occurs cotranslationally.
A) Schematic representation of cotranslational and post-translational modes of membrane protein integration into liposomes. B) Liposomes (final concentration of 10 mg/mL) were added to [35S]AAC translation reactions during translation or after the termination of protein synthesis as indicated, reactions were subjected to SGU, and fractions (1–4 from top to bottom, as indicated) were resolved by SDS-PAGE as in Fig. 1. C) Schematic of full length and ribosome-bound nascent chains analyzed in panels D and E. “AAC 249 RNC” and “AAC 316 RNC” intermediates have ribosome nascent chain lengths of 249 and 316 amino acids, respectively. “Tim9 87 RNC” has a chain length of 87 amino acids and is truncated just before the native stop codon. D) Ribosome-bound nascent chain constructs of AAC ([35S]AAC 249 and 316 intermediates) were translated in the absence (top panel) or presence (bottom panel) of liposomes and subjected to SGU fractionation as in panel B. E) Full length and ribosome-bound intermediates of AAC and Tim9 were translated in the presence of liposomes and subjected to SGU, and the top and bottom fractions of the gradient were resolved by SDS-PAGE.