Stbd1 is a protein of previously unknown function that is most prevalent in liver and muscle, the major sites for storage of the energy reserve glycogen. The protein is predicted to contain a hydrophobic N terminus and a C-terminal CBM20 glycan binding domain. Here, we show that Stbd1 binds to glycogen in vitro and that endogenous Stbd1 locates to perinuclear compartments in cultured mouse FL83B or Rat1 cells. When overexpressed in COSM9 cells, Stbd1 concentrated at enlarged perinuclear structures, co-localized with glycogen, the late endosomal/lysosomal marker LAMP1 and the autophagy protein GABARAPL1. Mutant Stbd1 lacking the N-terminal hydrophobic segment had a diffuse distribution throughout the cell. Point mutations in the CBM20 domain did not change the perinuclear localization of Stbd1, but glycogen was no longer concentrated in this compartment. Stable overexpression of glycogen synthase in Rat1WT4 cells resulted in accumulation of glycogen as massive perinuclear deposits, where a large fraction of the detectable Stbd1 co-localized. Starvation of Rat1WT4 cells for glucose resulted in dissipation of the massive glycogen stores into numerous and much smaller glycogen deposits that retained Stbd1. In vitro, in cells, and in animal models, Stbd1 consistently tracked with glycogen. We conclude that Stbd1 is involved in glycogen metabolism by binding to glycogen and anchoring it to membranes, thereby affecting its cellular localization and its intracellular trafficking to lysosomes.

Glycogen, a branched polymer of glucose, acts as an intracellular carbon and energy reserve in many tissues and cell types. An important pathway for its degradation is by transport to lysosomes in an autophagy-like process. It has been proposed that starch-binding domain-containing protein 1 (Stbd1) may participate in this mechanism by anchoring glycogen to intracellular membranes. In addition, Stbd1 has been reported to interact with a known autophagy protein, GABARAPL1, a member of the Atg8 family. Here, we confirm this interaction and identify an Atg8 interacting motif (AIM) in Stbd1 necessary for GABARAPL1 binding as judged by co-immunoprecipitation from cell extracts and co-localization in cells as evidenced by immunofluorescence microscopy. The AIM sequence of Stbd1 200HEEWEMV206 lies within a predicted disordered region of the molecule and fits the consensus of other AIM sequences in cargo-specifying proteins such as p62 and Nix. Mutation of the AIM, including single point mutations of either W203 or V206, eliminated the co-localization of Stbd1 with both over-expressed and endogenous GABARAPL1. Stbd1 may therefore function as a novel cargo binding protein that delivers glycogen to lysosomes in an autophagic pathway that could be termed “glycophagy”.

TREX1 is an endoplasmic reticulum (ER)-associated negative regulator of innate immunity. TREX1 mutations are associated with autoimmune and autoinflammatory diseases. Biallelic mutations abrogating DNase activity cause autoimmunity by allowing immunogenic self-DNA to accumulate, but it is unknown how dominant frame-shift (fs) mutations that encode DNase-active but mislocalized proteins cause disease. We found the TREX1 C-terminus suppressed immune activation by interacting with the ER oligosaccharyltransferase (OST) complex and stabilizing its catalytic integrity. C-terminal truncation of TREX1 by fs mutations dysregulated the OST complex, leading to free glycan release from dolichol carriers, as well as immune activation and autoantibody production. A connection between OST dysregulation and immune disorders was demonstrated in Trex1−/− mice, TREX1-V235fs patient lymphoblasts, and TREX1-V235fs knock-in mice. Inhibiting OST with aclacinomycin corrects the glycan and immune defects associated with Trex1-deficiency or fs mutation. This function of the TREX1 C-terminus suggests a potential therapeutic option for TREX1-fs mutant-associated diseases.

Since publication of this article, we have become aware of an error in the experimental protocol to visualize LAMP1 in cells by immunofluorescence. Specifically, the wrong antibodies were used, so assessments of LAMP1 distribution are not reliable. We did additional experiments, including analysis of the distribution of LAMP2, which is found in the same compartments as LAMP1. We observed no co-localization of LAMP2 and Stbd1. However, the primary conclusion of the study, the link between Stbd1 and glycogen metabolism, is unaffected, as is our hypothesis that Stbd1 anchors glycogen to membranes and may be involved in its localization and trafficking within the cell. The grant information footnote should read as follows. This work was supported, in whole or in part, by National Institutes of Health Grants R01 CA42755 and CA85804 (to C. W. D.), R01 AG031903 (to S. M.), T32 HL007147 (to S. S. and J. K. M.), and T32 GM007250 (to J. K. M.). The "Acknowledgments" should read as follows. The Dig2/RTP801 knock-out mice were obtained from Quark Pharmaceuticals, Inc., for whom they were exclusively generated at Lexicon. We thank Tamotsu Yoshimori and Noboru Mizushima for providing LC3 cDNA and Mark Jackson for suggestions regarding lentiviral shRNA. This work was supported in part by an Ohio Center for Innovative Immunosuppressive Therapeutics grant, which maintains the spinning disk confocal microscope in the Morphology Core Facility of the Department of Dermatology, Case Western Reserve University.THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 45, p. 39673, November 11, 2011 © 2011 ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.