TY - JOUR
T1 - Recruitment of Irgb6 to the membrane is a direct trigger for membrane deformation
AU - Yamada, Hiroshi
AU - Abe, Tadashi
AU - Nagaoka, Hikaru
AU - Takashima, Eizo
AU - Nitta, Ryo
AU - Yamamoto, Masahiro
AU - Takei, Kohji
N1 - Funding Information:
This work was supported, in part, by grants from the Ministry of Education, Science, Sports, and Culture of Japan (grant numbers 21K19484 to KT, 20K08591 to HY, and 22K06580 to TA), and by the Joint Usage/Research Center for Proteo-Interactome (PRiME), the Proteo-Science Center, Ehime University to HY. This work was supported by the Japan Agency for Medical Research and Development (AMED) (JP22wm0325010 to KT, MY, and RN).
Funding Information:
The work was supported by Okayama University Central Research Laboratory.
Publisher Copyright:
Copyright © 2022 Yamada, Abe, Nagaoka, Takashima, Nitta, Yamamoto and Takei.
PY - 2022/9/9
Y1 - 2022/9/9
N2 - Irgb6 is a member of interferon γ-induced immunity related GTPase (IRG), and one of twenty “effector” IRGs, which coordinately attack parasitophorous vacuole membrane (PVM), causing death of intracellular pathogen. Although Irgb6 plays a pivotal role as a pioneer in the process of PVM disruption, the direct effect of Irgb6 on membrane remained to be elucidated. Here, we utilized artificial lipid membranes to reconstitute Irgb6-membrane interaction in vitro, and revealed that Irgb6 directly deformed the membranes. Liposomes incubated with recombinant Irgb6 were drastically deformed generating massive tubular protrusions in the absence of guanine nucleotide, or with GMP-PNP. Liposome deformation was abolished by incubating with Irgb6-K275A/R371A, point mutations at membrane targeting residues. The membrane tubules generated by Irgb6 were mostly disappeared by the addition of GTP or GDP, which are caused by detachment of Irgb6 from membrane. Binding of Irgb6 to the membrane, which was reconstituted in vitro using lipid monolayer, was stimulated at GTP-bound state. Irgb6 GTPase activity was stimulated by the presence of liposomes more than eightfold. Irgb6 GTPase activity in the absence of membrane was also slightly stimulated, by lowering ionic strength, or by increasing protein concentration, indicating synergistic stimulation of the GTPase activity. These results suggest that membrane targeting of Irgb6 and resulting membrane deformation does not require GTP, but converting into GTP-bound state is crucial for detaching Irgb6 from the membrane, which might coincident with local membrane disruption.
AB - Irgb6 is a member of interferon γ-induced immunity related GTPase (IRG), and one of twenty “effector” IRGs, which coordinately attack parasitophorous vacuole membrane (PVM), causing death of intracellular pathogen. Although Irgb6 plays a pivotal role as a pioneer in the process of PVM disruption, the direct effect of Irgb6 on membrane remained to be elucidated. Here, we utilized artificial lipid membranes to reconstitute Irgb6-membrane interaction in vitro, and revealed that Irgb6 directly deformed the membranes. Liposomes incubated with recombinant Irgb6 were drastically deformed generating massive tubular protrusions in the absence of guanine nucleotide, or with GMP-PNP. Liposome deformation was abolished by incubating with Irgb6-K275A/R371A, point mutations at membrane targeting residues. The membrane tubules generated by Irgb6 were mostly disappeared by the addition of GTP or GDP, which are caused by detachment of Irgb6 from membrane. Binding of Irgb6 to the membrane, which was reconstituted in vitro using lipid monolayer, was stimulated at GTP-bound state. Irgb6 GTPase activity was stimulated by the presence of liposomes more than eightfold. Irgb6 GTPase activity in the absence of membrane was also slightly stimulated, by lowering ionic strength, or by increasing protein concentration, indicating synergistic stimulation of the GTPase activity. These results suggest that membrane targeting of Irgb6 and resulting membrane deformation does not require GTP, but converting into GTP-bound state is crucial for detaching Irgb6 from the membrane, which might coincident with local membrane disruption.
KW - GTPase
KW - IFN-inducible GTPase
KW - Irgb6
KW - membrane
KW - T. gondii
UR - http://www.scopus.com/inward/record.url?scp=85138636187&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85138636187&partnerID=8YFLogxK
U2 - 10.3389/fcimb.2022.992198
DO - 10.3389/fcimb.2022.992198
M3 - Article
C2 - 36159643
AN - SCOPUS:85138636187
SN - 2235-2988
VL - 12
JO - Frontiers in Cellular and Infection Microbiology
JF - Frontiers in Cellular and Infection Microbiology
M1 - 992198
ER -