SNARE proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here we used optical tweezers to observe in real time a long-sought SNARE assembly intermediate in which only the membrane-distal N-terminal half of the bundle is assembled. Our finding supports the zippering hypothesis, but suggests that zippering proceeds through three sequential binary switches, not continuously, in the N- and C-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force which mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 kBT to mediate fusion.

Synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins couple their stage-wise folding/assembly to rapid exocytosis of neurotransmitters in a Munc18-1-dependent manner. The functions of the different assembly stages in exocytosis and the role of Munc18-1 in SNARE assembly are not well understood. Using optical tweezers, we observed four distinct stages of assembly in SNARE N-terminal, middle, C-terminal, and linker domains (or NTD, MD, CTD, and LD, respectively). We found that SNARE layer mutations differentially affect SNARE assembly. Comparison of their effects on SNARE assembly and on exocytosis reveals that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion. Munc18-1 initiates SNARE assembly and structures t-SNARE C-terminus independent of syntaxin N-terminal regulatory domain (NRD) and stabilizes the half-zippered SNARE complex dependent upon the NRD. Our observations demonstrate distinct functions of SNARE domains whose assembly is intimately chaperoned by Munc18-1.DOI: http://dx.doi.org/10.7554/eLife.09580.001

SUMMARY While retrograde cargo selection in the Golgi is known to depend on specific signals, it is unknown whether anterograde cargo is sorted and anterograde signals have not been identified. We suggest here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterograde signal, and that it results in concentration in curved regions at the Golgi rims by simple physical chemistry. The rate of transport across the Golgi of two S-palmitoylated membrane proteins is controlled by Spalmitoylation. The bulk of S-palmitoylated proteins in the Golgi behave analogously, as revealed by click chemistry-based fluorescence and electron microscopy. These palmitoylated cargo concentrate in the most highly curved regions of the Golgi membranes, including the fenestrated perimeters of cisternae and associated vesicles. A palmitoylated transmembrane domain behaves similarly in model systems.

Recognition of DNA by proteins relies on direct interactions with specific DNA-functional groups, along with indirect effects that reflect variable energetics in the response of DNA sequences to twisting and bending distortions induced by proteins. Predicting indirect readout requires knowledge of the variations in DNA curvature and flexibility in the affected region, which we have determined for a series of DNA-binding sites for the E2 regulatory protein by using the cyclization kinetics method. We examined 16 sites containing different noncontacted spacer sequences, which vary by more than three orders of magnitude in binding affinity. For 15 of these sites, the variation in affinity was predicted within a factor of 3, by using experimental curvature and flexibility values and a statistical mechanical theory. The sole exception was traced to differential magnesium ion binding. Because many proteins deform DNA upon binding (1, 2), it is reasonable to expect that protein-DNA association might be facilitated by enhanced ease of DNA deformation and a match between intrinsic DNA shape in solution and the strained DNA conformation in the complex. The lack of a simple correspondence code between amino acids and DNA bases in hydrogen bonding, or direct readout, makes a general prediction of protein-DNA affinity impossible at this time. However, variations in contributions from indirect readout due to sequencedependent shape and mechanical properties may be predictable, yielding a partial structural code for protein-DNA interaction (3). Many attempts to test this idea have been performed during the last two decades with different proteins, such as nucleosome (4-6), cAMP-binding protein (7), 434 repressor (8, 9), TATA box-binding protein (10), and E2 protein (11).Among the impediments to realization of this objective is the difficulty of accurately determining the multiple DNA parameters involved, including magnitude and direction of curvature, helical twist, and bending and torsional flexibilities, for a variety of sequences corresponding to the region of indirect readout. The DNA cyclization method (1, 12, 13), in the high-throughput format we recently described (14), coupled with a statistical mechanical theory (15) for extracting the curvature and flexibility parameters from the data, provides a solution to this problem. Although the extent of variation of DNA flexibility with sequence remains controversial, recent results from the cyclization kinetics method show that a DNA sequence with high histone affinity (TATAAACGCC) has a nearly 2-fold smaller bending force constant and 35% less torsional rigidity than generic DNA (6). Also, an AT repeating sequence has 28% lower bending rigidity (14).Experimental testing of the accuracy of prediction of indirect readout requires that the nucleotides involved not be also engaged in direct interactions. A system that meets this requirement is the noncontacted spacer region in the DNA-binding site for the E2 protein encoded by the human papillomavirus (HPV) type 16 genome (11)....

Summary Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons—by orders of magnitude—than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging.