Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval

PLoS Biol. 2018 Jan 30;16(1):e2004411. doi: 10.1371/journal.pbio.2004411. eCollection 2018 Jan.

Abstract

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport / genetics*
  • Adaptor Proteins, Vesicular Transport / metabolism
  • Adaptor Proteins, Vesicular Transport / physiology*
  • CRISPR-Cas Systems
  • Endosomes / physiology
  • Golgi Apparatus / physiology
  • HeLa Cells
  • Humans
  • Lysosomes / genetics
  • Lysosomes / physiology
  • Mass Spectrometry
  • Membrane Proteins / metabolism
  • Phenotype
  • Protein Transport
  • Spastic Paraplegia, Hereditary / genetics
  • Vesicular Transport Proteins / metabolism

Substances

  • AP5B1 protein, human
  • Adaptor Proteins, Vesicular Transport
  • GOLIM4 protein, human
  • GOLM1 protein, human
  • Membrane Proteins
  • Vesicular Transport Proteins
  • sortilin