Starch-binding domain-containing protein 1 (Stbd1) and glycogen metabolism: Identification of the Atg8 family interacting motif (AIM) in Stbd1 required for interaction with GABARAPL1

Abstract

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 (200)HEEWEMV(206) 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".

Fig. 1

Interaction of GABARAPL1 with truncated mutants of Stbd1. Truncated hStbd1 lacking 1–24 residues or 262–358 residues with a C-terminal HA-tag (ΔN24-HA or Δ96-HA) and N-terminal Flag-tagged GABARAPL1 were expressed alone, or in the indicated combination in COS M9 cells. Control cells were transfected with empty pcDNA3 vector (Vector). (A) Schematic of potential AIM sites. (B) Immunoblotting of the cell lysates with the indicated antibody. Immunoprecipitation of GABARAPL1 with Anti-Flag antibodies covalently bound to agarose followed by immunoblotting with the indicated antibody.

Fig. 2

Interaction of GABARAPL1 with Stbd1 and potential Atg8 family interacting motif (AIM) mutants of Stbd1. hStbd1, full-length or mutants lacking 198–222 residues or double point mutations at potential AIMs with a C-terminal HA-tag (Δ198–222–HA (W203A, V206A)-HA, or (W212A, V215A)-HA) and N-terminal Flag-tagged GABARAPL1 were expressed alone, or in the indicated combination in COS M9 cells. Control cells were transfected with empty pcDNA3 vector (Vector). (A) Immunoblotting of the cell lysates with the indicated antibody (left panels). (B) Immunoprecipitation of GABARAPL1 with Anti-Flag antibodies covalently bound to agarose followed by immunoblotting with the indicated antibody (right panels).

Fig. 3

Subcellular localization of GABARAPL1 and Atg8 family interacting motif (AIM) mutants of Stbd1 co-expressed in COS M9 cells. Mutated hStbd1 with a C-terminal HA-tag was co-expressed in COS M9 cells with N-terminal Flag-tagged GABARAPL1 and immunostained with anti-HA antibodies (red) or anti-Flag antibodies (green). (A) Co-localization of hStbd1 and GABARAPL1 (merged in left panel) in cells co-expressing C-terminal HA-tagged full length hStbd1 (right panel) and N-terminal Flag-tagged GABARAPL1 (middle panel). (B) Loss of co-localization (merged in left panel) of Flag-tagged GABARAPL1 (middle panel) with potential AIM deletion mutant of hStbd1, Δ198–222–HA (right panel). (C) Impaired co-localization (merged in left panel) of Flag-tagged GABARAPL1 (middle panel) with double mutation in a potential AIM on hStbd1, (W203A, V206A)-HA (right panel). (D) Unaffected co-localization (merged in left panel) of Flag-tagged GABARAPL1 (middle panel) with double mutation in another potential AIM on hStbd1, (W212A, V215A)-HA (right panel). Nuclei were stained with Hoechst (blue). The scale bar is 20 µm.

Fig. 4

Subcellular localization of endogenous GABARAPL1 and overexpressed Stbd1 with Atg8 family interacting motif (AIM) mutations in COS M9 cells. Overexpressed full length or mutated hStbd1 with a C-terminal HA-tag and endogenous GABARAPL1 in COS M9 cells was immunostained with anti-HA antibodies (red) or anti-GABARAPL1 antibodies (green). (A) Co-localization of hStbd1 and GABARAPL1 (merged in left panel) in cells expressing C-terminal HA-tagged full length hStbd1 (right panel) and endogenous GABARAPL1 (middle panel). (B) Loss of co-localization (merged in left panel) of endogenous GABARAPL1 (middle panel) with potential AIM deletion mutant of hStbd1, Δ198–222–HA (right panel). (C) Impaired co-localization (merged in left panel) of endogenous GABARAPL1 (middle panel) with double mutation in potential AIM on hStbd1, (W203A, V206A)-HA (right panel). (D) Unaffected co-localization (merged in left panel) of endogenous GABARAPL1 (middle panel) with double mutation in another potential AIM on hStbd1, (W212A, V215A)-HA (right panel). Nuclei were stained with Hoechst (blue). The scale bar is 20 µm.