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.
Highlights
Adaptor protein (AP) complexes are a family of 5 evolutionarily ancient heterotetramers [1], which facilitate the transport of cargo from one membrane compartment of the cell to another
Knocking down the retromer complex exacerbated this phenotype. Both the cation-independent mannose 6-phosphate receptor (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
Understanding the function of AP-5 has been challenging. It is expressed at relatively low levels [4,5], and it has been lost from several model organisms, including Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae [6]. It is clearly important in humans, because mutations in its z subunit, encoded by the AP5Z1 gene, cause hereditary spastic paraplegia (HSP) [7], as do mutations in either SPG11 or SPG15 (SPG is an acronym for spastic paraplegia gene)
Summary
Adaptor protein (AP) complexes are a family of 5 evolutionarily ancient heterotetramers [1], which facilitate the transport of cargo from one membrane compartment of the cell to another. Understanding the function of AP-5 has been challenging It is expressed at relatively low levels (only about 10,000 copies in a HeLa cell, compared with about 300,000–1,000,000 copies for APs 1, 2, or 3) [4,5], and it has been lost from several model organisms, including Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae [6]. It is clearly important in humans, because mutations in its z subunit, encoded by the AP5Z1 gene (aka SPG48), cause hereditary spastic paraplegia (HSP) [7], as do mutations in either SPG11 or SPG15 (SPG is an acronym for spastic paraplegia gene). The disorder is classified as a complicated form of HSP, with various neurological abnormalities in addition to the typical degeneration of long corticospinal axons, which is a defining feature of all forms of HSP [8]
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