Autophagy targets intracellular molecules, damaged organelles, and invading pathogens for degradation in lysosomes. Recent studies have identified autophagy receptors that facilitate this process by ...binding to ubiquitinated targets, including NDP52. Here, we demonstrate that the small guanosine triphosphatase Rab35 directs NDP52 to the corresponding targets of multiple forms of autophagy. The active GTP-bound form of Rab35 accumulates on bacteria-containing endosomes, and Rab35 directly binds and recruits NDP52 to internalized bacteria. Additionally, Rab35 promotes interaction of NDP52 with ubiquitin. This process is inhibited by TBC1D10A, a GAP that inactivates Rab35, but stimulated by autophagic activation via TBK1 kinase, which associates with NDP52. Rab35, TBC1D10A, and TBK1 regulate NDP52 recruitment to damaged mitochondria and to autophagosomes to promote mitophagy and maturation of autophagosomes, respectively. We propose that Rab35-GTP is a critical regulator of autophagy through recruiting autophagy receptor NDP52. Synopsis GTPase Rab35 directs autophagy receptor NDP52 recruitment to ubiquitinated targets, thereby facilitating xenophagy, mitophagy and autophagosome maturation. GTP-bound active Rab35 accumulates on bacteria-containing endosomes and recruits NDP52 in a TBK1-dependent manner. Rab35-GTP directly interacts with NDP52 and facilitates its binding to ubiquitin to promote xenophagy. Rab35-mediated NDP52 recruitment to bacteria is inhibited by TBC1D10A through inactivation of Rab35. Rab35-mediated NDP52 targeting to damaged mitochondria and autophagosomes facilitates mitophagy and autophagosome maturation, respectively.
Abstract
Invading microbial pathogens can be eliminated selectively by xenophagy. Ubiquitin-mediated autophagy receptors are phosphorylated by TANK-binding kinase 1 (TBK1) and recruited to ...ubiquitinated bacteria to facilitate autophagosome formation during xenophagy, but the molecular mechanism underlying TBK1 activation in response to microbial infection is not clear. Here, we show that bacterial infection increases Ca
2+
levels to activate TBK1 for xenophagy via the Ca
2+
-binding protein TBC1 domain family member 9 (TBC1D9). Mechanistically, the ubiquitin-binding region (UBR) and Ca
2+
-binding motif of TBC1D9 mediate its binding with ubiquitin-positive bacteria, and TBC1D9 knockout suppresses TBK1 activation and subsequent recruitment of the ULK1 complex. Treatment with a Ca
2+
chelator impairs TBC1D9–ubiquitin interactions and TBK1 activation during xenophagy. TBC1D9 is also recruited to damaged mitochondria through its UBR and Ca
2+
-binding motif, and is required for TBK1 activation during mitophagy. These results indicate that TBC1D9 controls TBK1 activation during xenophagy and mitophagy through Ca
2+
-dependent ubiquitin-recognition.
Xenophagy, also known as antibacterial autophagy, plays a role in host defence against invading pathogens such as Group A Streptococcus (GAS) and Salmonella. In xenophagy, autophagy receptors are ...used in the recognition of invading pathogens and in autophagosome maturation and autolysosome formation. However, the mechanism by which autophagy receptors are regulated during bacterial infection remains poorly elucidated. In this study, we identified LAMTOR2 and LAMTOR1, also named p14 and p18, respectively, as previously unrecognised xenophagy regulators that modulate the autophagy receptor TAX1BP1 in response to GAS and Salmonella invasion. LAMTOR1 was localized to bacterium‐containing endosomes, and LAMTOR2 was recruited to bacterium‐containing damaged endosomes in a LAMTOR1‐dependent manner. LAMTOR2 was dispensable for the formation of autophagosomes targeting damaged membrane debris surrounding cytosolic bacteria, but it was critical for autolysosome formation, and LAMTOR2 interacted with the autophagy receptors NBR1, TAX1BP1, and p62 and was necessary for TAX1BP1 recruitment to pathogen‐containing autophagosomes. Notably, knockout of TAX1BP1 caused a reduction in autolysosome formation and subsequent bacterial degradation. Collectively, our findings demonstrated that the LAMTOR1/2 complex is required for recruiting TAX1BP1 to autophagosomes and thereby facilitating autolysosome formation during bacterial infection.
Xenophagy uses autophagy receptors, such as TAX1BP1, in the recognition of invading pathogens, in autophagosome maturation, and in autolysosome formation. However, the mechanism by which autophagy receptors are regulated during bacterial infection remains poorly elucidated. This study demonstrated that LAMTOR2 and LAMTOR1 are previously unrecognized xenophagy regulators that modulate the autophagy receptor TAX1BP1 in response to GAS and Salmonella invasion.
Rab35 GTPase recruits NDP52 to autophagy targets Minowa‐Nozawa, Atsuko; Nozawa, Takashi; Okamoto‐Furuta, Keiko ...
The EMBO journal,
15 September 2017, Letnik:
36, Številka:
18
Journal Article
Recenzirano
Odprti dostop
Autophagy targets intracellular molecules, damaged organelles, and invading pathogens for degradation in lysosomes. Recent studies have identified autophagy receptors that facilitate this process by ...binding to ubiquitinated targets, including NDP52. Here, we demonstrate that the small guanosine triphosphatase Rab35 directs NDP52 to the corresponding targets of multiple forms of autophagy. The active GTP‐bound form of Rab35 accumulates on bacteria‐containing endosomes, and Rab35 directly binds and recruits NDP52 to internalized bacteria. Additionally, Rab35 promotes interaction of NDP52 with ubiquitin. This process is inhibited by TBC1D10A, a GAP that inactivates Rab35, but stimulated by autophagic activation via TBK1 kinase, which associates with NDP52. Rab35, TBC1D10A, and TBK1 regulate NDP52 recruitment to damaged mitochondria and to autophagosomes to promote mitophagy and maturation of autophagosomes, respectively. We propose that Rab35‐GTP is a critical regulator of autophagy through recruiting autophagy receptor NDP52.
Synopsis
GTPase Rab35 directs autophagy receptor NDP52 recruitment to ubiquitinated targets, thereby facilitating xenophagy, mitophagy and autophagosome maturation.
GTP‐bound active Rab35 accumulates on bacteria‐containing endosomes and recruits NDP52 in a TBK1‐dependent manner.
Rab35‐GTP directly interacts with NDP52 and facilitates its binding to ubiquitin to promote xenophagy.
Rab35‐mediated NDP52 recruitment to bacteria is inhibited by TBC1D10A through inactivation of Rab35.
Rab35‐mediated NDP52 targeting to damaged mitochondria and autophagosomes facilitates mitophagy and autophagosome maturation, respectively.
GTPase Rab35 directs autophagy receptor NDP52 recruitment to ubiquitinated targets, thereby facilitating xenophagy, mitophagy and autophagosome maturation.
Abstract
Xenophagy, a type of selective autophagy, is a bactericidal membrane trafficking that targets cytosolic bacterial pathogens, but the membrane homeostatic system to cope with bacterial ...infection in xenophagy is not known. Here, we show that the endosomal sorting complexes required for transport (ESCRT) machinery is needed to maintain homeostasis of xenophagolysosomes damaged by a bacterial toxin, which is regulated through the TOM1L2–Rab41 pathway that recruits AAA-ATPase VPS4. We screened Rab GTPases and identified Rab41 as critical for maintaining the acidification of xenophagolysosomes. Confocal microscopy revealed that ESCRT components were recruited to the entire xenophagolysosome, and this recruitment was inhibited by intrabody expression against bacterial cytolysin, indicating that ESCRT targets xenophagolysosomes in response to a bacterial toxin. Rab41 translocates to damaged autophagic membranes via adaptor protein TOM1L2 and recruits VPS4 to complete ESCRT-mediated membrane repair in a unique GTPase-independent manner. Finally, we demonstrate that the TOM1L2–Rab41 pathway-mediated ESCRT is critical for the efficient clearance of bacteria through xenophagy.
Summary
Autophagy plays a crucial role in host defence by facilitating the degradation of invading bacteria such as Group A Streptococcus (GAS). GAS‐containing autophagosome‐like vacuoles (GcAVs) ...form when GAS‐targeting autophagic membranes entrap invading bacteria. However, the membrane origin and the precise molecular mechanism that underlies GcAV formation remain unclear. In this study, we found that Rab17 mediates the supply of membrane from recycling endosomes (REs) to GcAVs. We showed that GcAVs contain the RE marker transferrin receptor (TfR). Colocalization analyses demonstrated that Rab17 colocalized effectively with GcAV. Rab17 and TfR were visible as punctate structures attached to GcAVs and the Rab17‐positive dots were recruited to the GAS‐capturing membrane. Overexpression of Rab17 increased the TfR‐positive GcAV content, whereas expression of the dominant‐negative Rab17 form (Rab17 N132I) caused a decrease, thereby suggesting the involvement of Rab17 in RE–GcAV fusion. The efficiency of GcAV formation was lower in Rab17 N132I‐overexpressing cells. Furthermore, knockdown of Rabex‐5, the upstream activator of Rab17, reduced the GcAV formation efficiency. These results suggest that Rab17 and Rab17‐mediated REs are involved in GcAV formation. This newly identified function of Rab17 in supplying membrane from REs to GcAVs demonstrates that RE functions as a primary membrane source during antibacterial autophagy.
Invading bacteria can be degraded by selective autophagy, known as xenophagy. Recent studies have shown that the recruitment of autophagy adaptor proteins such as p62 to bacteria and its regulation ...by activated TANK-binding kinase 1 (TBK1) are required to overcome bacterial infection. However, the detailed molecular mechanisms behind this are not yet fully understood. Here, we show that the human guanylate-binding protein (GBP) family, especially GBP1, directs xenophagy against invading Group A Streptococcus (GAS) by promoting TBK1 phosphorylation. GBP1 exhibits a GAS-surrounding localization response to bacterially caused membrane damage mediated by the membrane damage sensor galectin-3. We found that GBP1 knockout attenuated TBK1 activation, followed by reduced p62 recruitment and lower bactericidal activity by xenophagy. Furthermore, GBP1-TBK1 interaction was detected by immunoprecipitation. Our findings collectively indicate that GBP1 contributes to GAS-targeted autophagy initiated by membrane damage detection by galectin-3 via TBK1 phosphorylation.
Xenophagy, also known as antibacterial autophagy, functions as a crucial defense system that can utilize intracellular pattern recognition sensors, such as NLRP4, to recognize and selectively ...eliminate bacterial pathogens. However, little is known about how NLRP4 regulates xenophagy. Here, we report that NLRP4 binds ARHGDIA (Rho GDP dissociation inhibitor α) to regulate Rho GTPase signaling and facilitate actin-mediated xenophagy. Specifically, NLRP4 is recruited to Group A Streptococcus (GAS) and colocalizes with GAS-containing autophagosome-like vacuoles (GcAVs), where it regulates ARHGDIA-Rho GTPase recruitment to promote autophagosome formation. The interaction between NLRP4, ARHGDIA, and Rho GTPases is regulated by ARHGDIA Tyr156 phosphorylation, which acts as a gate to induce Rho-mediated xenophagy. Moreover, ARHGDIA and Rho GTPase are involved in actin-mediated ATG9A recruitment to phagophores, facilitating elongation to form autophagosomes. Collectively, these findings demonstrate that NLRP4 functions as a Rho receptor complex to direct actin dynamics regulating xenophagy.
Macroautophagy/autophagy plays a critical role in immunity by directly degrading invading pathogens such as Group A Streptococcus (GAS), through a process that has been named xenophagy. We previously ...demonstrated that autophagic vacuoles directed against GAS, termed GAS-containing autophagosome-like vacuoles (GcAVs), use recycling endosomes (REs) as a membrane source. However, the precise molecular mechanism that facilitates the fusion between GcAVs and REs remains unclear. Here, we demonstrate that STX6 (syntaxin 6) is recruited to GcAVs and forms a complex with VTI1B and VAMP3 to regulate the GcAV-RE fusion that is required for xenophagy. STX6 targets the GcAV membrane through its tyrosine-based sorting motif and transmembrane domain, and localizes to TFRC (transferrin receptor)-positive punctate structures on GcAVs through its H2 SNARE domain. Knockdown and knockout experiments revealed that STX6 is required for the fusion between GcAVs and REs to promote clearance of intracellular GAS by autophagy. Moreover, VAMP3 and VTI1B interact with STX6 and localize on the TFRC-positive puncta on GcAVs, and are also involved in the RE-GcAV fusion. Furthermore, knockout of RABGEF1 impairs the RE-GcAV fusion and STX6-VAMP3 interaction. These findings demonstrate that RABGEF1 mediates RE fusion with GcAVs through the STX6-VAMP3-VTI1B complex, and reveal the SNARE dynamics involved in autophagosome formation in response to bacterial infection.
PDF
