Structure of Vps4 with circular peptides and implications for translocation of two polypeptide chains by AAA+ ATPases
- University of Utah School of Medicine, United States
- New York Structural Biology Center, United States
Abstract
Many AAA+ ATPases form hexamers that unfold protein substrates by translocating them through their central pore. Multiple structures have shown how a helical assembly of subunits binds a single strand of substrate, and indicate that translocation results from the ATP-driven movement of subunits from one end of the helical assembly to the other end. To understand how more complex substrates are bound and translocated, we demonstrated that linear and cyclic versions of peptides bind to the S. cerevisiae AAA+ ATPase Vps4 with similar affinities, and determined cryo-EM structures of cyclic peptide complexes. The peptides bind in a hairpin conformation, with one primary strand equivalent to the single chain peptide ligands, while the second strand returns through the translocation pore without making intimate contacts with Vps4. These observations indicate a general mechanism by which AAA+ ATPases may translocate a variety of substrates that include extended chains, hairpins, and crosslinked polypeptide chains.
Data availability
The refined model comprising the Vps4 ATPase domains of subunits A-E and the cyclic peptide is accessible via the PDB (RRID: SCR_012820; PDB ID: 6NDY) together with the 3.6 Å map from the combined dataset (RRID: SCR_003207, EMDB Accession Number EMD-0443). The complete model, including regions not subjected to atomic refinement such as the 12 Vta1VSL domains and subunit F, is also available via the PDB (PDB ID: 6OO2), together with the map containing Vta1VSL densities at all six Vps4 interfaces (RRID: SCR_003207, EMDB Accession Number EMD-20142). The two maps derived from the cF30 and cFF30 complex datasets individually, and the three maps for subunit F, have been deposited to the EMDB (RRID: SCR_ 003207, EMDB Accession Numbers EMD-20144, EMD-20147, EMD-20139, EMD-20140, EMD-20141).
-
Vps4 with Cyclic Peptide Bound in the Central PoreProtein Data Bank, 6NDY.
-
Vps4 with Cyclic Peptide Bound in the Central PoreProtein Data Bank, 6OO2.
-
Vps4 with Cyclic Peptide Bound in the Central PoreEMDataBank, EMD-20142.
Article and author information
Author details
Wesley I Sundquist
Christopher P Hill
Funding
National Institutes of Health (P50 GM082545)
- Han Han
- James M Fulcher
- Janet H Iwasa
- Michael S Kay
- Peter S Shen
- Christopher P Hill
National Institutes of Health (R01 GM112080)
- Wesley I Sundquist
National Institutes of Health (P41 GM103310)
- Venkata P Dandey
New York State Foundation for Science, Technology and Innovation
- Venkata P Dandey
Simons Foundation (SF349247)
- Venkata P Dandey
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- James M Berger, Johns Hopkins University School of Medicine, United States
Publication history
- Received: December 1, 2018
- Accepted: June 11, 2019
- Accepted Manuscript published: June 11, 2019 (version 1)
- Version of Record published: July 1, 2019 (version 2)
Copyright
© 2019, Han et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,836
- Page views
-
- 432
- Downloads
-
- 28
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Structure of Vps4 with circular peptides and implications for translocation of two polypeptide chains by AAA+ ATPases
eLife 8:e44071.
https://doi.org/10.7554/eLife.44071
Further reading
-
- Biochemistry and Chemical Biology
- Neuroscience
Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2–3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.
-
- Biochemistry and Chemical Biology
- Chromosomes and Gene Expression
N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.
