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Cell Cycle. 2016; 15(14): 1797–1798.
Published online 2016 April 22. doi:  10.1080/15384101.2016.1176400
PMCID: PMC4968891

TBC1D14 and TRAPP – Regulating autophagy through ATG9

Key requirements for cell homeostasis include the ability to remove and turn over unwanted or damaged proteins and organelles, and the ability to survive periods of stress such as nutrient deprivation. Eukaryotic cells achieve this through the autophagy pathways.1 Macroautophagy (which we refer to as autophagy here) is probably the best studied of these processes. It involves de novo formation of large double-membrane vesicles called autophagosomes, which enclose portions of cytosol and deliver them to the lysosome, where the contents are degraded and the components recycled back into the cytosol.

Autophagosome formation in response to stress is extremely rapid; in mammalian cells autophagosomes begin to form within 15 minutes of amino acid withdrawal. This requires a massive alteration in the cell’s membrane traffic to form these large membrane structures and direct them to the lysosome. We previously carried out an overexpression screen of TBC (Tre2, Bub2, Cdc16)-domain containing proteins – thought to be GAPs (GTPase-activating proteins) for the RAB family of small GTPases – and analyzed the effect on autophagy. Among 11 TBC proteins, we found TBC1D14 overexpression negatively regulates autophagy (Fig. 1A), via its effect on membrane traffic through the recycling endosome (RE). Other functions of the RE including transferrin recycling are also impaired (Fig. 1B). However, despite binding to RAB11, TBC1D14 acts as a RAB11 effector rather than a GAP, so how TBC1D14 exerted its effect on membrane traffic at the molecular level remained unclear.2

Figure 1.
TBC1D14 regulates RAB11-dependent trafficking of membrane from recycling endosomes to the growing phagophore (A), and transferrin recycling (B). TBC1D14 also acts via a mammalian TRAPPIII-like complex, returning ATG9 from RAB11-positive recycling endosomes ...

We studied the interactome of TBC1D14 using mass spectrometry, and identified subunits of the TRAPP (Trafficking Protein Particle) complex bound to TBC1D14.3 TRAPP complexes function as vesicle tethers, directing traffic from the ER and Golgi complex, and act as GEFs (GTP exchange factors) for RAB1. TRAPPIII in particular has also been shown to regulate autophagosome formation in yeast cells. In mammals, the picture is less clear, although a proteomics study in 2010 and data from a Salmonella xenophagy study have suggested TRAPP complexes support autophagy.

We confirmed the interaction between TBC1D14 and the TRAPP complex using co-immunoprecipitation approaches. We also found that TRAPP subunits, along with their GEF target RAB1, were on TBC1D14-induced RAB11-positive RE tubules. Our data supported a previously known link between RAB11-positive REs and RAB1-controlled Golgi compartments. We identified a 103 amino acid TRAPP binding region (TBR) in the N-terminus of TBC1D14, and found that overexpression of TBR inhibited autophagosome formation, and fragmented the Golgi complex. As TRAPP subunits have been implicated in secretory traffic and maintenance of Golgi integrity, we investigated this further and found that overexpression of TBR inhibited constitutive secretion.

Using proximity-based biotinylation combined with mass spectrometry, we identified the TRAPPC8 subunit of the mammalian TRAPP complex as mediating the interaction between TBC1D14 and TRAPP.3 Indeed, if TRAPPC8 is depleted, similar effects on autophagy are observed as when TBC1D14 is overexpressed. By studying TRAPPC8 interactors and using size-exclusion chromatography, we showed TRAPPC8 forms part of a mammalian TRAPPIII like complex. TRAPPC8 is an ortholog of the S. cerevisiae autophagy specific TRAPP subunit Trs85, and it has been shown Trs85 plays a role in autophagy by regulating ATG9 traffic and formation of autophagosomes.4,5 We investigated the trafficking of ATG9 in TRAPP-depleted and TBR-expressing HEK293 cells and found that in basal conditions ATG9, normally concentrated on Golgi membranes, was dispersed throughout the cytoplasm. This dispersion was independent of ULK1, the most upstream kinase regulating autophagy.3

Our findings suggest that TRAPP and TBC1D14 act together to regulate trafficking of ATG9 between RAB11-positive REs and RAB1-positive Golgi membranes (Fig. 1C). This trafficking is vital in ensuring a ready supply of ATG9 vesicles to support autophagosome formation, and also in maintaining Golgi integrity and functions including secretion. These findings are the first to characterize a role for mammalian TRAPP in autophagy, despite a known association between RAB1 activity and autophagosome formation.

The RE compartment has emerged as an important player in the regulation of the autophagic response. Sorting nexin 18 (SNX18) positively regulates traffic from the RE to the autophagosome.6 Additionally, recycling endosomes may act as a site from which ATG16-positive plasma membrane derived vesicles are recruited to the autophagosomes in a SNARE dependent manner.7 It will be particularly interesting to clarify the interplay between these 3 protein machineries in the modulation of autophagosome formation. It is possible that this endocytic trafficking machinery, including TBC1D14, Snx18 and the SNAREs, may act as a sensors of the nutrients in the cell’s environment, and TBC1D14 in particular may exploit its localization at the RE to modulate ATG9 traffic via the TRAPP complex in response to external nutrient status.

RE-localized TBC1D14 itself may play a broader role in cell function, by affecting the uptake of cargos such as the LDL receptor into the RE. Moreover, the contribution of RAB11-positive membranes to the cleavage furrow during cytokinesis could also be regulated by TBC1D14 (Fig. 1D), and a thorough characterization of the role of TBC1D14 in other aspects of cell function is warranted.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.


This work was supported by Cancer Research UK and the Francis Crick Institute which receives its core funding from Cancer Research UK, the UK Medical Research Council, and the Wellcome Trust.


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