Abstract
The key regulators of intracellular trafficking, Ypt/Rab GTPases, are stimulated by specific upstream activators and, when activated, recruit specific downstream effectors to mediate membrane-transport events. The yeast Ypt1 and its human functional homolog hRab1 regulate both endoplasmic reticulum (ER)-to-Golgi transport and autophagy. However, it is not clear whether the mechanism by which these GTPases regulate autophagy depends on their well-documented function in ER-to-Golgi transport. Here, we identify Atg11, the preautophagosomal structure (PAS) organizer, as a downstream effector of Ypt1 and show that the Ypt1-Atg11 interaction is required for PAS assembly under normal growth conditions. Moreover, we show that Ypt1 and Atg11 colocalize with Trs85, a Ypt1 activator subunit, and together they regulate selective autophagy. Finally, we show that Ypt1 and Trs85 interact on Atg9-containing membranes, which serve as a source for the membrane component of the PAS. Together our results define a Ypt/Rab module--comprising an activator, GTPase, and effector--that orchestrates the onset of selective autophagy, a process vital for cell homeostasis. Furthermore, because Atg11 does not play a role in ER-to-Golgi transport, we demonstrate here that Ypt/Rabs can regulate two independent membrane-transport processes by recruiting process-specific effectors.
Highlights
The key regulators of intracellular trafficking, Ypt/Rab GTPases, are stimulated by specific upstream activators and, when activated, recruit specific downstream effectors to mediate membrane-transport events
Ypt1 is required for endoplasmic reticulum (ER)-toGolgi transport [3,4,5], and the TRAPP I complex acts as its guanine nucleotide exchange factors” (GEFs) [6, 7]
We verified this interaction in both plasmid orientations (Fig. 1 B and C) and showed that it is specific to Ypt1, because Atg11 does not interact with Ypt6, Ypt31, or Sec4 (Fig. 1B); the last two play a role in autophagy [20]
Summary
Atg, which interacts with multiple Atg proteins through three of its coiled-coil (CC) domains (Fig. 1A) [19], was identified as a Ypt interactor in two independent yeast-two hybrid screens. CC 2 and 3 of Atg are required and sufficient for the interaction with Ypt1-GTP (Fig. 1B and Fig. S1) These results suggest that Atg is a Ypt effector, with the middle region of Atg mediating the interaction. This region is involved in multiple Atg interactions and is required for its function in selective autophagy [19].
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