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Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation

Nature Structural & Molecular Biology volume 23, pages 821–829 (2016)Cite this article

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Abstract

Hsp70s use ATP hydrolysis to disrupt protein-protein associations and to move macromolecules. One example is the Hsc70- mediated disassembly of the clathrin coats that form on vesicles during endocytosis. Here, we exploited the exceptional features of these coats to test three models—Brownian ratchet, power-stroke and entropic pulling—proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models and instead support a collision-pressure mechanism whereby collisions between clathrin-coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also found that self-association augments collision pressure, thereby allowing disassembly of clathrin lattices that have been predicted to be resistant to disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates.

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Figure 1: Structural context of approaches to test Hsc70 disassembly mechanisms.
Figure 2: Moving the Hsc70-binding site slows disassembly and reveals a reaction intermediate of large scattering amplitude, and replacing it with a FLAG tag allows disassembly by anti-FLAG Fabs.
Figure 3: Hsc70 binding to cages under conditions that block disassembly leads to a massive increase in light-scattering.
Figure 4: Cryo-EM reconstructions of cages with or without Hsc70ΔC or Hsc70.
Figure 5: Scattering increases due to Hsc70 binding reflect binding of multiple Hsc70s per CHC, not cage expansion.
Figure 6: Accumulation of Hsc70 associated with cages drives cage disassembly, and Hsc70 self-association augments its cage disassembly force.
Figure 7: Hsc70 binding makes cages less compressible but more prone to catastrophic deformations.
Figure 8: General model for the Hsp70 mechanochemical cycle.

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Acknowledgements

We thank L. Wang of UTHSCSA for technical assistance; T. Kirchhausen of HMS for CHC cDNA; C. Brantner and D. Sackett of NICHD, the NHLBI EM Core Facility for help with EM at an early stage of the work; and D. Sackett and R. Nossal at NICHD, D. Luque from ISCIII and J. Stachowiak from UT-Austin for helpful discussions. This work was supported by NS029051 (to E.M.L.) and GM118933 (to E.M.L. and R.S.); the NIH Intramural Research Program (NIBIB); and by grants BFU2013-44202 from the Spanish Ministry of Economy and Innovation and S2013/MIT-2807 from the Madrid Regional Government to J.M.V. H.-S.L. was supported in part by a scholarship from the Taiwan National Science Council (NSC103-2917-I-564-072).

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  1. Hsien-Shun Liao

    Present address: Present address: Department of Mechanical Engineering, National Taiwan University, Taipei, Taiwan.,

Authors and Affiliations

  1. Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

    Rui Sousa, Suping Jin & Eileen M Lafer

  2. Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA

    Hsien-Shun Liao & Albert J Jin

  3. Department for Macromolecular Structures, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain.,

    Jorge Cuéllar & José M Valpuesta

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Contributions

R.S. designed and performed experiments, analyzed data and wrote the paper. H.-S.L., J.C., S.J., A.J.J. and E.M.L. designed and performed experiments, and analyzed data. J.M.V. designed experiments and analyzed data.

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Integrated supplementary information

Supplementary Figure 1 Standardization, specificity and controls for experiments measuring disassembly by light-scattering.

a: Standard curves for scattering by various concentrations of either clathrin cages (black squares) or triskelia (red circles) expressed as concentration of CHC. Error bars show +/- SD for 6 (cages) or 13 (triskelia) repetitions of one experiment each. Across the concentration ranges used in our experiments the amount of light scattering by either cages or triskelia is linear (R2 for both fits >0.99). Panels b-e: Removal of auxilin, ATP, Hsc70 ATP binding or hydrolysis activity, or deletion of the Hsc70 NBD greatly slows or abrogates Hsc70 mediated cage disassembly. b: Blue trace shows a complete reaction with 2 μM Hsc70 as in fig. 2A. Black trace shows an identical reaction with ATP omitted. Red trace shows a reaction in which the Hsc70 was replaced with 2 μM PBD (missing the NBD). c. Blue trace as in b; black=auxilin omitted; red=ATP and auxilin omitted; c: Blue as in a; black=ATPγS replaces ATP; red= ADP replaces ATP. Experimental: All reactions in b-d were carried out on the same day with the same set of cages, with each reaction condition repeated 4-11 times (trace thickness corresponds to +S.E.). The blue trace is identical in all plots but data for different reaction conditions was plotted separately for clarity as the reaction profiles otherwise overlap. e: Blue trace is complete reaction with WT Hsc70; red trace is with Hsc70 mutant that cannot bind ATP (G201E/G202D; Morgan, J.R. et al. A role for an Hsp70 nucleotide exchange factor in the regulation of synaptic vesicle endocytosis. J Neurosci 33, 8009 (2013)); black trace is for ATP hydrolysis deficient mutant (E175S; Jiang, J. et al. Structural basis of J cochaperone binding and regulation of Hsp70. Mol Cell 28, 422 (2007)). Trace thickness shows +/- s.e. for 8-15 repetitions of 1-3 independent experiments. Panels f, g: Rapid disassembly of cages in which the canonical Hsc70 binding site is moved is not due to Hsc70 binding at non-canonical sites. f: Light scattering vs. time plotted for disassembly reactions with 2 μM Hsc70 and either cages in which the Hsc70 binding site was moved by 25AA (red; average of 11 replicates with trace thickness giving +/- s.e.) or in which it was eliminated (black; average of 9 replicates with +25AA FLAG tag cages). g: As in f, but using 2 μM Hsc70ΔC (averages of 7 and 10 replicates, respectively). The residual Hsc70-mediated disassembly seen with cages in which the Hsc70 site is replaced with a FLAG-tag appears to be primarily due to Hsc70 binding weakly to other sites on the tails, as we found that complete deletion of the tails markedly slowed disassembly even further than simply removing the Hsc70 binding site by mutation (data not shown, available on request). h: Anti-FLAG Fabs can’t dissociate WT cages. WT cages reacted with 2 μM Hsc70 (black trace; 5 replicates averaged) or 2 μM Fab (red trace; 7 replicates averaged). i: Auxilin stabilizes clathrin cage against disassembly. +10AA FLAG-tag Clathrin cages with auxilin in 1.3x excess of CHC were reacted with anti-FLAG Fab as described in fig. 2K (black trace), or in the absence of auxilin (red trace). Effects on auxilin on cage stability and contributions to disassembly are difficult to parse as it also makes an essential contribution to disassembly by recruiting the Hsc70 to the cage. By using FLAG-tag cages and anti-FLAG Fab we can remove the recruitment contribution and measure just auxilin’s contribution to cage stability. It is seen that the Fab disassemble the FLAG-tag cages more slowly in the presence of auxilin than without it, indicating that auxilin binding stabilizes cages and makes them more resistant to disassembly.

Supplementary Figure 2 Tight coupling limits fitting of microscopic reaction parameters from ensemble experiments that measure disassembly by light-scattering.

A: Fit (magenta) of a reaction profile (blue) for one experiment using the ‘Simplefit’ script (Supplementary Information). The obtained values of the variables (ka=Hsc70 binding rate; kd=cage disassembly rate; A1=scattering amplitude of the transient Hsc70:cage complex) were used to calculate the overall rate of disassembly (kov) and the time to 50% reaction complete (t1/2) according to the equation: kov=1/(1/([Hsc70]*ka)+1/kd). B: As in A, but kd was fixed at 2x the value in A. Note the large differences in ka and A1 between panels A and B, but the very similar value for kov and t1/2 for both fits. While the fit in A is better, both fits are quite good. This is because a reaction characterized by “fast Hsc70 binding (ka), slow disassembly (kd) and a small scattering amplitude for the transient cage:Hsc70 complex (A1)” will generate a profile very similar to a “slow binding, fast disassembly, large transient scattering amplitude increase” reaction. In practice, we found that experimental variation was greater than the differences in the quality of fits shown above so that microscopic rate parameters like ka, kd or A1 could not be accurately determined from such data. However, the kov values were relatively insensitive to this coupling.

Supplementary Figure 3 Bivalent anti-FLAG antibodies are ineffective at disassembling FLAG-tag cages.

A. Light scattering vs. time in disassembly reactions with +0AA FLAG cages run as in fig. 2J but with the indicated concentrations of anti-FLAG IgG rather than Fab. Traces are averages from 7-9 repetitions. B: The same data as in A, but plotted with error bars giving +/- s.e. range for each experiment (error bars removed in A for clarity due to data overlap). C. Plot of the 1st 15 seconds of the reactions to better resolve the initial antibody binding step. Note that the amplitude of the scattering increase (~40%) is similar to that seen with Fab (fig. 2J-K), and is therefore consistent with bivalent binding of the antibody since monovalent binding would result in binding of similar numbers of Ab and Fab molecules and with a larger scattering increase for the former due to its larger MW. D: Gel analysis confirms that anti-FLAG IgG binds cages bivalently. Plot of IgG/CHC ratios vs. [IgG] (at 1, 3 and 9 μM; errors are +/- s.e. for n=3) shows that 0.45-0.65 IgG molecules per CHC bind to cages, while 0.8-1.2 Fab molecules per CHC are seen to bind in identical experiments shown in figure 5.

Supplementary Figure 4 The large Hsc70-induced increase in scattering at pH 6.0 is not due to cage aggregation, because the amplitude and rate of the scattering increase is insensitive to cage concentration.

Hsc70 could induce large increases in scattering by causing cages to aggregate. Since aggregation would be at least a 2nd-order reaction, its rate should be sensitive to cage concentration. A. Scattering vs. time in pH 6.0 disassembly reactions with [Hsc70] at 2 μM and clathrin cages at the indicated concentrations (expressed in molar concentration of CHC with data plotted representing averages +s.e. of 10, 19, 15 and 14 replicates at 38, 75, 150 and 300 nM, respectively) and with scattering normalized so that the starting level in the reaction with 150 nM cages is 1.0. B. Data from A replotted so that the starting level of scattering in all the reactions is normalized to 1.0.

Supplementary Figure 5 Binding of large numbers of Hsc70s per CHC in pH 6–stabilized cages requires ATP, auxilin and an Hsc70-binding site in the CHC tails.

Plot of the # of Hsc70s per CHC pelleting with cages in absence of ATP, auxilin or an Hsc70 binding site (FLAG-tag cages; error is +se for 2 analyses of one experiment). Residual Hsc70 binding to cages in which the Hsc70 binding site is replaced with a FLAG-tag appears to be primarily due to Hsc70 binding weakly to other sites on the tails, as we found that complete deletion of the tails reduced binding (as assessed by the amount of 70 that pellets with cages) even more (to a level not meaningfully different than seen in the absence of cages; data not shown, available on request) than does altering the Hsc70 binding site by mutation. The nature of the 'non-specific' sites at which 70's bind to such cages is unknown, but such binding is consistent with the known promiscuous binding specificity of Hsc70 and the conclusion (Bocking, T., Aguet, F., Harrison, S.C. & Kirchhausen, T. Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat Struct Mol Biol 18, 295-301 (2011)), that, even with WT cages, approximately half of the Hsc70 molecules that associate with cages bind at 'non-specific' sites that are ineffective for disassembly.

Supplementary Figure 6 Hsc70ΔC competes for Hsc70 binding to cages and inhibits cage disassembly by Hsc70 at low pH.

Reduced disassembly activity and scattering increases with Hsc70ΔC vs. Hsc70 could be due to weaker binding of Hsc70ΔC to cages, rather than reduced self-association. If so then adding excess Hsc70ΔC to reactions with Hsc70 shouldn't inhibit disassembly by the latter since Hsc70ΔC shouldn't effectively compete with Hsc70 for cage binding. Conversely, if Hsc70ΔC and Hsc70 bind cages with similar affinity, but Hsc70ΔC doesn't disassemble as effectively once bound, then Hsc70ΔC should compete with and inhibit Hsc70 disassembly. To test this we carried out disassembly at pH 6.3 and 1 μM Hsc70, conditions under which disassembly is slow and depends on Hsc70 self-association. A. Cage disassembly reactions carried out at pH 6.3 with 1 μM Hsc70 and with 0 (black; average of 5 replicates), 3 μM (red; average of 8 replicates with trace thickness showing +/- se), or 9 μM (blue; average of 8 replicates) Hsc70ΔCterm. Hsc70ΔCterm at 3- and 9x excess over Hsc70 is observed to reduce disassembly rates by ~2x and ~4x, respectively. B. SDS PAGE analysis of pellet fractions of 1mM Hsc70, 0.23 μM Auxilin, 0.15 μM (lanes 1-6) or 0 μM (lanes 7-12) cages (concentrations of cages expressed as [CHC]) and with Hsc70ΔC at 1 (lanes 2, 8), 2 (lanes 3, 9), 4 (lanes 4, 10), 8 (lanes 5, 11) or 16 (lanes 6, 12) μM. Hsc70ΔC reduces the amount of Hsc70 that pellets with the cages. This experiment was independently performed 2x with similar results.

Supplementary Figure 7 Hsc70, but not Hsc70ΔC, disassembles cages and forms filamentous oligomers at pH 6.0.

Cages were mixed with ATP and either Hsc70ΔC or Hsc70 as described for cryo-EM, but then incubated for 30 minutes to allow disassembly to occur and then negatively stained for EM. In the Hsc70ΔC reaction, cages were still present against a background of excess Hsc70ΔC (panel A). In reactions with Hsc70 almost no cages could be seen (panel B), though clearly obvious triskelia were not visible because the spindly triskelia are likely obscured by Hsc70 molecules in 10-fold molar excess of CHC. Long Hsc70 oligomers were abundant (a higher magnification view of these is shown in panel C), with each unit of the oligomer estimated as large enough to accommodate 1-2 Hsc70 molecules. In panels A and B a model of a reconstructed cage is inset to provide a scale marker.

Supplementary Figure 8 AFM analysis of clathrin cages.

A. Left panel: QNM AFM at 4nm pixel resolution and 100 pN tip force of clathrin-auxilin cages and under buffer (pH 6.0) reveals characteristic polygonal surface structure. Top right panel: 3D height view of a field of clathrin-auxilin cages. Lower right panel: Nanoscale intra-cage compressibility profiles of the same field. The shared X-Y scale bar of 100 nm and Z-color bar representation are for the height (100 nm) and the compression (30 nm). The white threshold lines in the right panels demarcate 35 nm height level around each observed cage to identify cage tops for the computation of the observed maximal and mean compression for each cage. B. QNM AFM of fields of cages with the indicated associated proteins. The top panel plots cage height while the lower panel plots compression (shared Z scale of 100 and 50 nm for height & compression, respectively). Note the wide distribution of compression profiles in the +Hsc70 panel where cages display both very large and small compressions, while the distribution of compression sizes in the other panels is more uniform. C. Height distributions of cages with the indicated associated proteins (200 pN tip force). D. Left panel: Distribution of mean and maximum compression values for cages with the indicated proteins (200 pN tip force). Mean compression corresponds the average of the compression observed when each cage was sampled with either a 3x3 or 4x4 grid (9 or 16 measurements respectively, depending on cage size). Maximum compression corresponds to the largest compression measured during AFM probing of each cage.

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Supplementary Figures 1–8, Supplementary Tables 1–3 and Supplementary Notes 1 and 2 (PDF 2014 kb)

Supplementary Data Sets 1–3

Primer sequences, uncropped gel images and cryo-EM fields. (PDF 550 kb)

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Sousa, R., Liao, HS., Cuéllar, J. et al. Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation. Nat Struct Mol Biol 23, 821–829 (2016). https://doi.org/10.1038/nsmb.3272

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