Molecular machinery of membrane fusion with single-molecule measurements

Abstract

Most biological processes such as DNA replication, transcription, translation, cell adhesion, protein folding, protein and nucleic acid unfolding, protein degradation, and membrane fusion are operated systematically by molecular machinery, especially proteins. To reveal the molecular mechanism of the machinery, it is inevi-table to observe and manipulate molecules at the molecular level. We studied the molecular machinery for membrane fusion using single molecule measurement techniques such as electron microscopy, single-molecule fluorescence, and single-molecule force spectroscopy which have been developed and will be de-veloped by biophysicists in the future. Here, we show the molecular mechanism of the assembly and disas-sembly of the SNARE complex, the key molecular machinery for membrane fusion. First of all, we observed the intermediates of membrane fusion using cryo-transmission electron microscopy (cryo-TEM) and single-vesicle fluorescence during yeast SNARE complex formation. We could directly visualize two kinds of intermediates - one is two bilayers are on the contact part of two vesicles (two-stacked bilayers state) and the other is the state that one bilayer is on the contact part between two vesicles (hemifusion diaphragm). Moreover, we observed that incorporating 35 % PE into vesicles can increase hemi-fusion diaphragm populations. Therefore, we conclude the following: 1) Vesicle fusion intermediates can be captured in cryo-TEM samples. In addition, we can observe hemifusion diaphragm which has been observed using other methods. 2) The geometry of PE lipids can stabilize hemifusion diaphragm. We also directly visu-alized fusion pore opening, which is initiated from an edge of the vertex ring (where boundary and outside membrane meet on docked vesicles) and the residual membrane was one bilayer. Therefore, we concluded that hemifusion diaphragm might not be a nonproductive end state of the fusion reaction. Secondly, we reconstituted and characterized NSF/$\alpha$ -SNAP mediated SNARE complex disassem-bly using single molecule fluorescence methods and single molecule force spectroscopy. The results are as follows: 1) NSF uses one-round ATP turnover to disassemble SNARE complex. 2) $\alpha$ -SNAP not only tightly connects NSF to the SNARE complex, but also destabilizes the C-terminal part of the SNARE complex to support the action of NSF. 3) the SNARE complex is disassembled in a single burst within 20 ms by NSF. These show that the molecular mechanism of NSF is different from other AAA+ ATPases such as helicase or protein unfoldase which are known to progressively unwind substrates. Our results show how an AAA+ ATPase can induce global unwinding of a substrate protein complex, which turns out to be a much more effi-cient mechanism than processive unwinding.