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Related Experiment Video

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Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers
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Cargo selectivity of yeast sorting nexins.

Björn D M Bean1, Michael Davey1, Elizabeth Conibear1

  • 1Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, Child and Family Research Institute, University of British Columbia, Vancouver, Canada.

Traffic (Copenhagen, Denmark)
|November 25, 2016
PubMed
Summary

Sorting nexins are key proteins in membrane trafficking. This study identified new cargo proteins for sorting nexins in yeast, revealing novel functions and sorting motifs.

Keywords:
Ear1EndosomeGolgiHigh-Throughput MicroscopyRetrograde TraffickingRetromerSnx3Snx4Sorting NexinYeastYmd8Ymr010wYmr253c

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Sorting nexins (SNXs) are proteins containing a PX domain, involved in phospholipid binding and membrane trafficking.
  • Their precise functions and cargo specificities remain largely uncharacterized for many SNXs.
  • Understanding SNX functions is crucial for deciphering cellular cargo selection and transport.

Purpose of the Study:

  • To identify novel sorting nexin cargo using a high-throughput imaging screen in Saccharomyces cerevisiae.
  • To investigate the roles of specific sorting nexins, including Snx4 and the retromer complex, in protein localization.
  • To elucidate sorting motifs and conserved residues involved in retromer and Snx3-dependent sorting.

Main Methods:

  • A high-throughput imaging screen was employed to assess the localization of GFP-tagged membrane proteins.
  • Deletions of nine different sorting nexins were screened for protein mislocalization.
  • Functional assays and analysis of chimeric reporters were used to identify sorting motifs.

Main Results:

  • 27 proteins were identified that require at least one sorting nexin for correct localization.
  • 23 of these identified proteins represent novel sorting nexin cargo.
  • Specific cytosolic domains and conserved residues in Ear1, Ymd8, and Ymr010w were found to mediate retromer-dependent sorting.

Conclusions:

  • This study significantly expands the known cargo of sorting nexins in yeast.
  • Novel sorting motifs and conserved residues critical for retromer and Snx3-mediated transport were discovered.
  • The findings provide a consensus sequence for retromer and Snx3-dependent sorting, advancing our understanding of membrane trafficking regulation.