Rab GTPases as “eat me” signals for selective autophagy

Autophagy is a highly conserved cellular degradation pathway wherein cytoplasmic components are enclosed within double-membrane autophagosomes and subsequently transported to lysosomes for breakdown and recycling (1, 2). This process not only provides an internal source of nutrients under stress conditions, such as nutrient starvation and hypoxia, but also plays a crucial role in cellular quality control by removing defective organelles and misfolded proteins (3).

While autophagy was initially considered a nonselective pathway, research from the past decades has highlighted the extensive presence of selective autophagy, which ensures the precise removal of damaged or excess organelles, protein aggregates, and invading pathogens, thereby maintaining cellular homeostasis and function (4). Almost all organelles have been reported to undergo selective autophagy. The corresponding processes are named with organelle-specific prefixes: mitophagy for mitochondria, lipophagy for lipid droplets, xenophagy for exogenous pathogens, etc.

A central challenge in selective autophagy lies in cargo recognition—how cells precisely identify and label specific targets for degradation. Current findings have revealed a complex network of molecular mechanisms involved in cargo recognition. Cargo receptors often contain cargo recognition domains and LC3-interacting regions that facilitate the recruitment of LC3/GABARAP proteins on the autophagosome membrane, facilitating the engulfment of cargos into autophagosomes (4). Beyond receptors, additional signals are necessary for the selective removal of unwanted materials while sparing essential components. Ubiquitination commonly marks cargoes for degradation. Additionally, phosphorylation and damage-induced exposure of lipids/sugars on targets are also involved in selective autophagy (5).

Rab GTPases are a large family of small GTP-binding proteins that play crucial roles in orchestrating the complex processes of vesicular transport within eukaryotic cells (6). Although Rab GTPases are traditionally acknowledged for their involvement in nonselective autophagy through diverse mechanisms (7), recent findings indicate that they might also play a direct role in selective autophagy (8, 9). However, the precise mechanism is largely unexplored. Here, Zhao et al. systematically analyzed the involvement of Rab GTPases in selective autophagy and revealed that they engage with specific cargo receptors for precise spatiotemporal regulation of selective autophagy (10).

The study commences by observing a progressive decline of endogenous Rab2 levels in cultured mammalian cells upon starvation, with restoration upon lysosomal inhibition, mirroring the behavior of the autophagy substrate p62/SQSTM1. Through GFP cleavage and tandem fluorescence reporter assays, the authors confirmed the lysosomal degradation of Rab2. Rab proteins undergo cycling between an active GTP-bound state and an inactive GDP-bound state. Further mutational analysis revealed that the GDP-bound inactive form of Rab2 or its prenylation-defective mutant failed to undergo degradation, indicating a dependency on membrane-anchoring ability.

To expand the scope of their investigation, Zhao et al. screened a panel of human Rab GTPases using GFP cleavage assays. They identified that 31 out of 45 Rab GTPases undergo lysosomal degradation. Subsequent screens in Atg7 knockout cells and microscopic analysis of tandem mCherry-GFP–tagged Rab GTPases confirmed the involvement of canonical autophagy in the degradation of 25 Rab proteins. A series of experiments was performed to further support these findings. For example, purified lysosomes contained multiple Rab proteins that were resistant to protease treatment. Through GFP-tagging and knockout experiments, a similar pattern of autophagic degradation of Rab GTPases was validated in Saccharomyces cerevisiae and Caenorhabditis elegans, providing robust evidence for the evolutionary conservation of Rab GTPase degradation via autophagy.

To explore the underlying mechanisms, Zhao et al. systematically examined the interaction between Rab GTPases undergoing autophagic degradation and 18 reported selective autophagy receptors. Most receptors, both cytosolic and membrane-bound, interacted with at least one Rab GTPase. Notably, NDP52, a key player in mitophagy and xenophagy, as well as TOLLIP, an aggrephagy receptor, exhibited binding to multiple Rab GTPases. Most of the autophagy-degraded Rab GTPases also interacted with at least one receptor, emphasizing the crucial role of these interactions in the autophagy process. Biochemical investigations further demonstrated direct binding of specific Rab GTPases with NDP52 in vitro through its ZN motif. The extensive association of Rab GTPases with autophagy receptors underscores their crucial role in signal propagation.

The above findings prompted the authors to investigate the functional role of Rab GTPases using selective autophagy models. They first demonstrated that specific Rab GTPases were degraded upon mitophagy induction, and the degradation relied on intact autophagy. Subsequent experiments revealed that mitochondrial stress conditions triggered the mitochondrial targeting of multiple Rab GTPases, with Rab8 as the most prominent one. Through various approaches, including immuno-electron microscopy and live-cell imaging, they confirmed Rab8’s localization on the mitochondrial outer membrane. Interestingly, the mitochondrial targeting of Rab GTPases for mitophagy was independent of the canonical Parkin–PINK1 pathway but dependent on prenylation of the proteins. Further investigations in C. elegans and yeast supported the role of Rab GTPases as positive regulators of mitophagy. Similar mechanisms were also verified in lipophagy and xenophagy. These findings support the notion that the model is common in different types of selective autophagy and conserved among various organisms.

What mechanisms regulate the function of Rab GTPases as signals for selective autophagy? Zhao et al. focused on Rab regulators, including RabGGTase, Rab GDP dissociation inhibitor (GDI), and LRRK2. They found that RabGGTase plays a positive role in this signaling process by promoting Rab prenylation, which is crucial for autolysosomal degradation of Rab8. Knockdown of RabGGTase components reduced Rab8 degradation. In contrast, GDI binds to Rabs to prevent their membrane association. Inhibiting GDIs facilitated the degradation of Rab2, Rab8, and Rab9. Additionally, suppression of the activity of LRRK2, a Parkinson’s disease–associated kinase that may phosphorylate and inhibit certain Rab GTPases, affected their interactions with autophagy receptors and autophagic degradation. These results suggest a complex regulatory network involving RabGGTase, Rab GDI, and LRRK2 in modulating the activity of Rab GTPases as key “autophagy cues” in selective autophagy processes.

In conclusion, the findings of Zhao et al. (10) reveal a novel function of Rab GTPase in regulating selective autophagy by intricate interplay with autophagy receptors (Fig. 1). This inspiring model sparks a cascade of intriguing questions: How do Rab GTPases collaborate with known receptors to facilitate selective autophagy? Does cargo recognition by Rab GTPase also play a role in nonselective autophagy? How do cells distinguish between healthy organelles decorated with Rabs and damaged membranes destined for degradation? How do cells discern and attach Rab GTPase tags to damaged membranes while preserving functional organelles? Elucidating these molecular mechanisms will not only advance our understanding of autophagy regulation but will also pave the way for potential therapeutic interventions targeting these pathways in various disease contexts.