Immunity-related GTPase M (IRGM) Proteins Influence the Localization of Guanylate-binding Protein 2 (GBP2) by Modulating Macroautophagy

Maria K Traver,Stanley C Henry,Viviana Cantillana,Tim Oliver,Julia P Hunn,Jonathan C Howard,Sandra Beer,Klaus Pfeffer,Jörn Coers,Gregory A Taylor

doi:10.1074/jbc.m111.251967

Abstract

The immunity-related GTPases (IRGs) are a family of proteins induced by interferon-γ that play a crucial role in innate resistance to intracellular pathogens. The M subfamily of IRG proteins (IRGM) plays a profound role in this context, in part because of the ability of its members to regulate the localization and expression of other IRG proteins. We present here evidence that IRGM proteins affect the localization of the guanylate-binding proteins (GBPs), a second family of interferon-induced GTP-binding proteins that also function in innate immunity. Absence of Irgm1 or Irgm3 led to accumulation of Gbp2 in intracellular compartments that were positive for both the macroautophagy (hereafter referred to as autophagy) marker LC3 and the autophagic adapter molecule p62/Sqstm1. Gbp2 was similarly relocalized in cells in which autophagy was impaired because of the absence of Atg5. Both in Atg5- and IRGM-deficient cells, the IRG protein Irga6 relocalized to the same compartments as Gbp2, raising the possibility of a common regulatory mechanism. However, other data indicated that Irga6, but not Gbp2, was ubiquitinated in IRGM-deficient cells. Similarly, coimmunoprecipitation studies indicated that although Irgm3 did interact directly with Irgb6, it did not interact with Gbp2. Collectively, these data suggest that IRGM proteins indirectly modulate the localization of GBPs through a distinct mechanism from that through which they regulate IRG protein localization. Further, these results suggest that a core function of IRGM proteins is to regulate autophagic flux, which influences the localization of GBPs and possibly other factors that instruct cell-autonomous immune resistance.

Highlights

The Absence of Irgm1 or Irgm3 Affects the Localization of Gbp2—As mentioned above, we and others have previously demonstrated that the GMS immunity-related GTPases (IRGs) proteins Irgm1 and Irgm3 regulate the localization of GKS IRG proteins
We addressed whether the IRGM/ GMS proteins affect broader processes that may affect the functioning of other proteins, including the guanylate binding proteins (GBPs)
We have further shown that IRGM protein deficiency does not alter Gbp2 ubiquitination, it apparently alters Lys-63-linked polyubiquitination of Irga6, which localizes to Gbp2-positive autophagosomal structures

Summary

To whom correspondence should be addressed

IRG2 proteins share a number of biochemical functions: they are GTPases [5, 6], they bind lipid membranes in various intracellular membrane compartments (6 – 8), and they form dimers and/or oligomers [5, 9]. These and other characteristics relate them to the dynamins, a large family of GTPbinding proteins that are involved in vesicle formation, vesicle trafficking, and other aspects of lipid membrane remodeling (10 –12). Our findings suggest that IRGM proteins may have activities that extend beyond the IRG protein family to influence other IFN-␥-induced effectors by modulating autophagy

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION