Assisted and Unassisted Protein Insertion into Liposomes

Abstract

The insertion of newly synthesized membrane proteins is a well-regulated and fascinating process occurring in every living cell. Several translocases and insertases have been found in prokaryotic and eukaryotic cells, the Sec61 complex and the Get complex in the endoplasmic reticulum and the SecYEG complex and YidC in bacteria and archaea. In mitochondria, TOM and TIM complexes transport nuclear-encoded proteins, whereas the Oxa1 is required for the insertion of mitochondria-encoded membrane proteins. Related to the bacterial YidC and the mitochondrial Oxa1 are the Alb3 and Alb4 proteins in chloroplasts. These membrane insertases are comparably simple and can be studied in vitro, after their biochemical purification and reconstitution in artificial lipid bilayers such as liposomes or nanodiscs. Here, we describe the recent progress to study the molecular mechanism of YidC-dependent and unassisted membrane insertion at the single molecule level.

Figure 1

Membrane potential across liposomes. An electrochemical potential was generated by adding 0.25 μM valinomycin (left arrow) to liposomes with 200 mM Na⁺ inside and 200 mM K⁺ outside measured by oxonol VI fluorescence (A). Alamethicin was added as a control, which forms a nonselective cation peptide pore and therefore leads to the collapse of the membrane potential (right arrow). No potential was measured when the liposomes had inside and outside 200 mM Na⁺ (B). When the liposomes were generated from insufficiently purified lipids and treated as in (A), the initial potential steadily decreased (C), indicating leaky bilayers (9, 29).

Figure 2

Dosing the electrochemical membrane potential. Inside/outside K⁺ concentrations were set to 20 μM/200 mM K⁺ (A), 200 μM/200 mM K⁺ (B), 2 mM/200 mM K⁺μM (C), and 40 mM/200 mM K⁺ (D). From the apparent oxonol VI fluorescence and the calculated Nernst potential, Δψ was determined as −240 mV (A), −177 mV (B), and 74−50 mV (C).

Figure 3

Topology analysis of YidC in the reconstituted proteoliposomes. Purified YidC protein was mixed with DOPC lipids and unilamellar proteoliposomes were generated with an extruder. The topology of YidC can be analyzed by protease accessibility by adding trypsin (A) (9). The proteolytic fragments were detected using immunoblotting by either an antiserum directed to the large periplasmic domain (left panel) or to the cytoplasmic tail (right panel). The topology of YidC modified with a fluorophore can be determined by the relative amount that is protected from the quencher added to the outside of the proteoliposome (B). The periplasmic domain of YidC was labeled with Atto520. For both cases (A and B), >80% of the YidC periplasmic domain was found in the liposomal lumen in the case of DOPC-reconstituted YidC.

Figure 4

Insertion of Pf3 (A) or KcsA (B) in DOPC liposomes in the absence or presence of YidC, measured by FCS. The proteins were purified and labeled with Atto520, then added to DOPC liposomes (black) or to YidC proteoliposomes (red). The fluorescence was measured for 10 min in the presence of 100 mM KI. Whereas the insertion of KcsA was independent of the YidC insertase, the Pf3 coat protein strictly requires YidC.

Figure 5

Loss of insertion competence of Pf3 coat protein after incubation in aqueous media in the absence of YidC-proteoliposomes. Low nanomolar concentrations of Pf3 were added to the buffer, followed by the addition of proteoliposomes after defined time points (1, 2, 5, or 15 min) or simultaneously (0 min), displayed on the x axis. Immediately after the proteoliposomes have been added, relative rates of insertion were tested by determining the number of bursts, according to Fig. 4, during 6.5 min measurements (y axis).

Figure 6

Insertion of single protein molecules into YidC proteoliposomes observed by FCS-based FRET. Atto520-labeled Pf3 protein was added to a solution containing Atto647N-labeled YidC, reconstituted into proteoliposomes. (A) The high temporal resolution of distance measurements between fluorophores, determined by FRET, allows resolving the individual events taking place on a molecular level, as illustrated in (B). The protein Pf3 (red) freely diffuses in solution (a), until it encounters a YidC-containing proteoliposome and binds to the insertase (green, b). The distance between the fluorophores decreases, which demonstrates a close interaction between YidC and Pf3 during the membrane insertion process (c). The increasing distance between donor and acceptor fluorophor and finally the loss of a FRET signal indicate the release of Pf3 from the insertase YidC (d, adapted from (13)). To see this figure in color, go online.