Putative fusogenic activity of NSF is restricted to a lipid mixture whose coalescence is also triggered by other factors

Abstract

It has recently been reported that N-ethylmaleimide-sensitive fusion ATPase (NSF) can fuse protein-free liposomes containing substantial amounts of 1,2-dioleoylphosphatidylserine (DOPS) and 1, 2-dioleoyl-phosphatidyl-ethanolamine (DOPE) (Otter-Nilsson et al., 1999). The authors impart physiological significance to this observation and propose to re-conceptualize the general role of NSF in fusion processes. We can confirm that isolated NSF can fuse liposomes of the specified composition. However, this activity of NSF is resistant to inactivation by N-ethylmaleimide and does not depend on the presence of alpha-SNAP (soluble NSF-attachment protein). Moreover, under the same conditions, either alpha-SNAP, other proteins apparently unrelated to vesicular transport (glyceraldehyde-3-phosphate dehydrogenase or lactic dehydrogenase) or even 3 mM magnesium ions can also cause lipid mixing. In contrast, neither NSF nor the other proteins nor magnesium had any significant fusogenic activity with liposomes composed of a biologically occurring mixture of lipids. A straightforward explanation is that the lipid composition chosen as optimal for NSF favors non-specific fusion because it is physically unstable when formed into liposomes. A variety of minor perturbations could then trigger coalescence.

Fig. 1. NSF-mediated liposome–liposome fusion is restricted to specific lipid compositions. Donor and acceptor liposomes with the lipid compositions indicated were mixed at a molar ratio of 1:5 in a cuvette at 37°C (for details see Materials and methods). Donor liposomes were supplemented with rhodamine- and NBD-labeled DPPE (0.8 mol% each). NSF was added to a final concentration of 5 μg/ml at the time indicated. After 10 min the reaction was terminated by adding Triton X–100 at a final concentration of 0.1% (w/v). Data are expressed as a percentage of maximal NBD fluorescence after addition of detergent.

Fig. 2. SNARE-mediated membrane fusion does not depend on a specific lipid composition. SNARE-containing liposomes of the lipid compositions indicated were reconstituted as described previously (Weber et al., 1998), see also Materials and methods. (A) POPC:DOPS (85:15; donor and acceptor); (B) DOPE:DOPS (50:50; donor) and DOPE:DOPC (50:50; acceptor); (C) ‘Golgi’ lipids (donor) and POPC:DOPS (85:15; acceptor). Lipid recovery and efficiency of protein reconstitution were found to be similar under all conditions. v– and t–SNARE-containing liposomes were mixed on ice and incubated at 37°C for the times indicated. Where indicated, the cytosolic domain of VAMP was added at a final concentration of 0.4 mg/ml. Fluorometric recordings were obtained as described previously (Weber et al., 1998).

Fig. 3. Factors mediating fusion of DOPE:DOPS donor and DOPE:DOPC acceptor liposomes. Donor and acceptor liposomes were mixed with various reagents and fusion was followed by the increase of NBD fluorescence and analyzed as described in the legend to Figure 1. (A) Donor (DOPE:DOPS; 50:50) and acceptor (DOPE:DOPC; 50:50) liposomes were premixed. NSF (blue curve), α–SNAP (red curve), glyceraldehyde-3–phosphate dehydrogenase (GA-3-P-DH, black curve) or lactic dehydrogenase (LDH, green curve) were added at a final concentration of 5 μg/ml. (B) NSF (blue curve), α–SNAP (red curve), glyceraldehyde-3-phosphate dehydrogenase (GA-3-P-DH, black curve) or lactic dehydrogenase (LDH, green curve) were added to premixed ‘Golgi’-like donor and acceptor liposomes at a final concentration of 5 μg/ml. (C) MgCl₂ was added at the concentration indicated to a premix of DOPE:DOPS (50:50) donor and DOPE:DOPC (50:50) acceptor liposomes (black curve, 3 mM; blue curve, 6 mM). (D) MgCl₂ (final concentration of 6 mM) was added to either ‘Golgi’ lipid donor and acceptor liposomes (blue curve) or POPC:DOPS (85:15) donor and acceptor liposomes (black curve).

Fig. 4. Fusion of liposomes is not dependent on the ATPase activity of NSF. (A) NSF-mediated fusion of liposomes is not inhibited by NEM. NSF was pre-incubated with 2 mM NEM for 30 min on ice followed by the addition of DTT (final concentration of 3 mM) to quench free NEM. A control sample was incubated for 30 min on ice with NEM that had been pre-incubated with DTT. NEM-treated NSF (red curve) or control NSF (blue curve) was added to a premix of DOPE:DOPS (50:50) donor and DOPE:DOPC (50:50) acceptor liposomes and lipid mixing was monitored. (B) NSF-mediated fusion of liposomes is not inhibited by EDTA. NSF was added to a premix of DOPE:DOPS (50:50) donor and DOPE:DOPC (50:50) acceptor liposomes in fusion buffer containing 1 mM MgCl₂, 0.5 mM ATP, 10 IU/ml creatine kinase and 10 mM creatine phosphate as ATP-regenerating system, in the presence (red curve) or absence (blue curve) of 5 mM EDTA. (C) Nucleotide requirements for NSF-mediated fusion. NSF-bound nucleotide was exchanged against either ADP, ATP or ATPγS as described in Materials and methods. Exchange efficiency was monitored employing [α–³³P]ATP and found to be >90% for each condition. A control sample of NSF was not subjected to nucleotide exchange. NSF in the presence of the various nucleotides was added to premixed liposomes and fusion monitored as described above.