Academic journal article Genetics

Trs33-Containing TRAPP IV: A Novel Autophagy-Specific Ypt1 GEF

Academic journal article Genetics

Trs33-Containing TRAPP IV: A Novel Autophagy-Specific Ypt1 GEF

Article excerpt

IN autophagy, cargo destined for degradation is engulfed by the double-membrane autophagosomes (APs), and is shuttled to the lysosome. Depending on the cargo and the growth conditions, autophagy can be generic or selective (Nair and Klionsky 2005; Nakatogawa et al. 2009). All autophagy pathways start with the formation of the preautophagosomal structure (PAS), which is comprised of the autophagy-specific proteins Atgs and membrane (Weidberg et al. 2011). Like all other intracellular trafficking pathways, autophagy is regulated by the conserved Ypt/Rab GTPases (Ao et al. 2014). When stimulated by guanine-nucleotide exchange factors (GEFs), Ypt/Rabs bind their downstream effectors, which include intracellular trafficking machinery components, like motors and tethers (Segev 2001). Recently, a role for Ypt/Rabs in coordination of intracellular trafficking steps and pathways has been proposed (Segev 2011; Lipatova et al. 2015).

Inyeast, three Ypts regulate the different steps of autophagy: Ypt1 is required for the beginning ofautophagy, PAS formation (Lipatova et al. 2012), while Vps21 and Ypt7 play a role in later steps that lead to the fusion of APs with the vacuole (the yeast lysosome) (Wang et al. 2002; Chen et al. 2014). Ypt31/32 were also implicated in autophagy, but the step is not clear (Zou et al. 2013). The established role of Ypt1 is the regulation of ER-to-Golgi transport (Segev 1991), and that of Vps21 and Ypt7 is in endocytosis (Schimmoller and Riezman 1993; Singer-Kruger et al. 1994). Interestingly, whereas both Vps21 and Ypt7 function in autophagy and endocytosis in the context of the same GEF-GTPase-effector modules (Wang et al. 2002; Chen et al. 2014), Ypt1 does not. Instead, two different TRAPP complexes, TRAPP I and TRAPP III, stimulate Ypt1 in the secretory and autophagy pathways, respectively (Lipatova et al. 2015). Likewise, in secretion and autophagy Ypt1 interacts with different effectors; e.g., Atg11 is an autophagy-specific effector of Ypt1 (Lipatova et al 2012).

Currently, three TRAPP complexes are known: I, II, and III, and theirmultiple subunits are conserved fromyeastto human cells (Kim et al 2016). TRAPP I contains four core essential subunits, and TRAPP III contains the nonessential Trs85 in addition to core-TRAPP and the Trs20 adaptor. Both TRAPP I and TRAPP III act as Ypt1 GEFs (Morozova et al 2006; Cai et al 2008; Lynch-Day et al 2010). TRAPP II contains two essential large subunits, Trs120 and Trs130, the nonessential subunits Trs65 and Trs33, the Trs20 adaptor, and core-TRAPP. TRAPP II localizes to trans-Golgi, and, while still controversial, acts as a GEF for Ypt31/32 (Kim et al. 2016). A role for a third nonessential subunit, Trs33, was shown in the assembly of TRAPP II only in the absence of Trs65 (Tokarev et al. 2009) (Figure 1A).

Ypt1 is essential for autophagy based on the fact that the autophagy phenotypes of autophagy-specificyptl mutations are as severe as those of core-atg deletions (Lynch-Day et al. 2010; Lipatova et al. 2012). In contrast, whereas Trs85 plays a role in autophagy, it is not essential for this process (Lipatova et al. 2012). The question that drove this research project is why Ypt1 is essential to autophagy whereas its autophagy-specific GEF is not. Here, we show that Trs33 plays a role in autophagy, and together with Trs85 is required for Ypt1-mediated PAS formation. Based on results presented here, we propose the existence of a new TRAPP complex, the Trs33-containing TRAPP IV, which together with TRAPP III activates Ypt1 in the onset of autophagy. Because all players are conserved from yeast to human cells, we propose that the human homologs of Trs33, TrappC6A and B, regulate Rab1-mediated autophagy.

Materials and Methods

Strains, plasmids, and reagents

Strains used in this paper are summarized in Supplemental Material, Table S1. Plasmids used in this study are summarized in Table S2. All chemical reagents were purchased from Fisher Scientific (Hampton, NH), except for the following: Nitrogen bases were purchased from US Biological (Swampscott, MA); ProtoGel for Western blots from National Diagnostics (Atlanta, GA); Bacto peptone and Bacto agar from BD Difco (Franklin Lakes, NJ); salmon testes DNA, amino acids, p-nitrophenyl phosphate, and protease inhibitors from Sigma (St. …

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