The Uso1 globular head interacts with SNAREs to maintain viability even in the absence of the coiled-coil domain

  1. Ignacio Bravo-Plaza
  2. Victor G Tagua
  3. Herbert N Arst Jr
  4. Ana Alonso
  5. Mario Pinar
  6. Begoña Monterroso
  7. Antonio Galindo
  8. Miguel A Peñalva  Is a corresponding author
  1. Centro de Investigaciones Biológicas, Spain
  2. Universidad de La Laguna, Spain
  3. Imperial College London, United Kingdom
  4. MRC Laboratory of Molecular Biology, United Kingdom

Abstract

Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Genetic studies placed Uso1 paradoxically acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S in the GHD that rescued lethality of rab1-deficient Aspergillus nidulans. The mutations are phenotypically additive, their combination suppressing the complete absence of RAB1, which emphasizes the key physiological role of the GHD. In living hyphae Uso1 recurs on puncta (60 sec half-life) colocalizing partially with the Golgi markers RAB1, Sed5 and GeaA/Gea1/Gea2, and totally with the retrograde cargo receptor Rer1, consistent with Uso1 dwelling in a very early Golgi compartment from which ER residents reaching the Golgi recycled back to the ER. Localization of Uso1, but not of Uso1E6K/G540S, to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. That Uso1 delocalization correlates with a decrease in the number of Gea1 cisternae supports that Uso1-and-Rer1-containing puncta are where the protein exerts its physiological role. In S-tag-coprecipitation experiments Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Using purified proteins, we show that Bos1 and Bet1 bind the Uso1 GHD directly. However, Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. G540S alone markedly increases GHD binding to Bos1, whereas E6K causes a weaker effect, correlating with their phenotypic contributions. AlphaFold2 predicts that G540S increases binding of the GHD to the Bos1 Habc domain. In contrast, E6K lies in an N-terminal, potentially alpha-helical, region that sensitive genetic tests indicate as required for full Uso1 function. Remarkably, this region is at the end of the GHD basket opposite to the end predicted to interact with Bos1. We show that unlike dimeric full-length and CTR∆ Uso1 proteins, the GHD lacking the CC/CTR dimerization domain, whether originating from bacteria or Aspergillus extracts and irrespective of whether it carries or not E6K/G540S, would appear to be monomeric. With the finding that overexpression of E6K/G540S and wild-type GHD complement uso1∆, our data indicate that the GHD monomer is capable of providing, at least partially, the essential Uso1 functions, and that long-range tethering activity is dispensable. Rather, these findings strongly suggest that the essential role of Uso1 involves the regulation of SNAREs.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 8 through 11, 13 -figure supplement 2 and 14Supplemental Table II and III(strains and primers) ensure the reproducibility of the experiments

Article and author information

Author details

  1. Ignacio Bravo-Plaza

    Department of Cellular and Molecular, Centro de Investigaciones Biológicas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0934-9084
  2. Victor G Tagua

    Área de Medicina Preventiva y Salud Pública, Universidad de La Laguna, San Cristóbal de La Laguna, Islas Canarias, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1494-6895
  3. Herbert N Arst Jr

    Department of Infectious Diseases, Imperial College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Ana Alonso

    Department of Cellular and Molecular, Centro de Investigaciones Biológicas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
  5. Mario Pinar

    Department of Cellular and Molecular, Centro de Investigaciones Biológicas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2415-8721
  6. Begoña Monterroso

    Department of Structural and Chemical Biology, Centro de Investigaciones Biológicas, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2538-084X
  7. Antonio Galindo

    Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. Miguel A Peñalva

    Department of Cellular and Molecular, Centro de Investigaciones Biológicas, Madrid, Spain
    For correspondence
    penalva@cib.csic.es
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3102-2806

Funding

Agencia Estatal de Investigación (predoctoral contract)

  • Ignacio Bravo-Plaza

Agencia Estatal de Investigación (Grant)

  • Miguel A Peñalva

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2023, Bravo-Plaza 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

  • 990
    views
  • 124
    downloads
  • 7
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

Share this article

https://doi.org/10.7554/eLife.85079

Further reading

    1. Cell Biology
    2. Evolutionary Biology
    Paul Richard J Yulo, Nicolas Desprat ... Heather L Hendrickson
    Research Article

    Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.

    1. Cell Biology
    2. Developmental Biology
    Pavan K Nayak, Arul Subramanian, Thomas F Schilling
    Research Article

    Mechanical forces play a critical role in tendon development and function, influencing cell behavior through mechanotransduction signaling pathways and subsequent extracellular matrix (ECM) remodeling. Here we investigate the molecular mechanisms by which tenocytes in developing zebrafish embryos respond to muscle contraction forces during the onset of swimming and cranial muscle activity. Using genome-wide bulk RNA sequencing of FAC-sorted tenocytes we identify novel tenocyte markers and genes involved in tendon mechanotransduction. Embryonic tendons show dramatic changes in expression of matrix remodeling associated 5b (mxra5b), matrilin1 (matn1), and the transcription factor kruppel-like factor 2a (klf2a), as muscles start to contract. Using embryos paralyzed either by loss of muscle contractility or neuromuscular stimulation we confirm that muscle contractile forces influence the spatial and temporal expression patterns of all three genes. Quantification of these gene expression changes across tenocytes at multiple tendon entheses and myotendinous junctions reveals that their responses depend on force intensity, duration and tissue stiffness. These force-dependent feedback mechanisms in tendons, particularly in the ECM, have important implications for improved treatments of tendon injuries and atrophy.