Abstract
ABSTRACTThe sorting nexins (SNXs) are a family of peripheral membrane proteins that direct protein trafficking decisions within the endocytic network. Emerging evidence in yeast and mammalian cells implicates a subgroup of SNXs in selective and non-selective forms of autophagy. Using siRNA and CRISPR-Cas9, we demonstrate that the SNX-BAR protein SNX4 is needed for efficient LC3 (also known as MAP1LC3) lipidation and autophagosome assembly in mammalian cells. SNX-BARs exist as homo- and hetero-dimers, and we show that SNX4 forms functional heterodimers with either SNX7 or SNX30 that associate with tubulovesicular endocytic membranes. Detailed image-based analysis during the early stages of autophagosome assembly reveals that SNX4–SNX7 is an autophagy-specific SNX-BAR heterodimer, required for efficient recruitment and/or retention of core autophagy regulators at the nascent isolation membrane. SNX4 partially colocalises with juxtanuclear ATG9A-positive membranes, with our data linking the autophagy defect upon SNX4 disruption to the mis-trafficking and/or retention of ATG9A in the Golgi region. Taken together, our findings show that the SNX4–SNX7 heterodimer coordinates ATG9A trafficking within the endocytic network to establish productive autophagosome assembly sites, thus extending knowledge of SNXs as positive regulators of autophagy.
Highlights
Macroautophagy describes the sequestration of cytoplasmic material within double membranebound vesicles that deliver their contents to lysosomes for degradation and recycling
As a Bin/ Amphiphysin/Rvs (BAR) domain protein with affinity for PtdIns(3)P, we explored the possibility that SNX4 influences autophagy via its association with autophagosomes and/or PtdIns(3)P-enriched autophagosome assembly sites
Transient expression of mCherry– SNX4 in stable GFP–LC3B HEK293 cells revealed that whilst the majority of SNX4-positive membrane structures were spatially separated from GFP–LC3B puncta, a minor fraction could be found in close proximity to GFP–LC3B-labelled autophagosomes (Fig. 1C)
Summary
Macroautophagy ( referred to as autophagy) describes the sequestration of cytoplasmic material within double membranebound vesicles (autophagosomes) that deliver their contents to lysosomes for degradation and recycling. Molecules with affinity for either PtdIns(3)P and/or curved membrane profiles [for example, Bin/ Amphiphysin/Rvs (BAR) domain-containing proteins (McMahon and Boucrot, 2015)] have been implicated in the control of autophagosome biogenesis. Acting during the autophagosome expansion phase is the BAR domain-containing protein, SH3GLB1 ( known as BIF-1 or Endophilin B1), which binds to UVRAG–Beclin-1 to stimulate the autophagy PIK3C3 kinase ( known as VPS34), whilst facilitating trafficking of ATG9 to the autophagosome assembly site (Takahashi et al, 2007, 2011). Identification of further PtdIns(3)P effectors and/or BAR domain-containing proteins with the potential to influence autophagosome expansion and shaping remains a key objective
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