Biochemical and Biophysical Research Communications
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
Highlights
► Starch binding domain protein 1 (Stbd1) binds glycogen and membranes. ► Stbd1 interacts with GABARAPL1 and co-localizes in cells via a single Atg8 interacting motif (AIM). ► The AIM is 200HEEWEMV206.
Introduction
Glycogen, a branched glucose polymer, acts as an intracellular carbon and energy reserve in many cell types [1], [2]. Many studies of glycogen metabolism have focused on its cytosolic synthesis and degradation. Although glycogen is usually considered cytosolic, glycogen particles have been found by electronic microscopy close to intracellular membranes such as the endoplasmic reticulum (ER) in liver [3] and the sarcoplasmic reticulum (SR) in muscle [4]. Glycogen is also present in the lysosomes of mammalian cells where it is directly hydrolyzed by lysosomal acid α-glucosidase (acid maltase, GAA) [5]. The significance of the lysosomal hydrolysis pathway is underscored by the fact that defects in the glucosidase cause a severe glycogen storage disease, Pompe disease, which eventually destroys tissues by over-accumulating glycogen in lysosomes [6].
The molecular mechanism for glycogen trafficking to the lysosome is not well understood although autophagy or an autophagy-like process is likely to be involved [7], [8]. Initial clues for a mechanism came from a recent study of starch-binding domain-containing protein 1 (Stbd1), also known as genethonin 1 [9]. Stbd1 contains a conserved N-terminal hydrophobic sequence and a conserved C-terminal carbohydrate binding module of the CBM20 family (see Supplemental Fig. 2 of Jiang et al. [7]). The intervening region within Stbd1, which is overall much less conserved [7], is predicted to be disordered by analysis of the Stbd1 sequence using the “predictors of natural disordered regions” (PONDR) algorithm [10]. Stbd1 binds glycogen in vitro and is associated with glycogen in cells [7]. Jiang et al. [7] proposed that Stbd1 functions to anchor glycogen to membranes via its N-terminus. A further linkage to vesicular transport of glycogen is suggested by a yeast two-hybrid screen using human Stbd1 lacking the first 171 residues as bait. Among the targets identified were two autophagy related proteins, GABARAP and GABARAPL1, which are members of the Atg8 family [11]. Because GABARAPL1 more strictly co-distributed with Stbd1 in cells, it may be the preferred physiological binding partner. GABARAPL1 was originally cloned as an estrogen-regulated message in guinea pig endometrial glandular epithelial cells (GEC) and given the name Gec1 [12] which is still in use. Several functions have been proposed for GABARAPL1, including a role in autophagy, but much remains to be learned about its physiological function [13].
In mammalian cells, macroautophagy was initially thought to be an essentially random process for recycling cellular materials in response to nutritional deprivation [14], [15], [16], [17], [18]. However, there is emerging evidence that autophagic pathways can be more selective [17], [18]. The autophagic disposal of several organelles has been described by processes named pexophagy (peroxisomes), mitophagy (mitochondria), ribophagy (ribsosomes) and reticulophagy (surplus edoplasmic reticulum). Aggrephagy describes removal of protein inclusions called aggrosomes and lipophagy refers to the disposal of oxidized lipids. Xenophagy is a process to eliminate intracellular pathogens like bacteria and viruses [16], [17], [18]. One idea to explain such selectivity is that cargo-specific receptors are coupled to individual selective autophagic pathways. Several cargo adaptor proteins have been identified, including p62, NBR1 and Nix. The ubiquitin-binding protein p62, also called sequestosome protein-1 (SQSTM1), is proposed to function as a cargo receptor for autophagic degradation of ubiquitinated protein substrates [17], [19], [20]. NBR1 has been suggested to function as an SQSTM1/p62 partner in disposal of misfolded proteins [21]. Nix has been proposed to selectively target the clearance of mitochondria in a ubiquitin-independent manner [22]. These receptors are all proposed to act by binding to Atg8 family members via short, specific and conserved sequences in the cargo receptors, termed Atg8-family interacting motifs (AIM) [23] or LC3 interacting regions (LIR) [20]. We propose that Stbd1 acts as a cargo receptor for glycogen and report the identification of the AIM in Stbd1 responsible for its interaction with GABARAPL1.
Section snippets
Plasmid construction
Mammalian expression vectors containing HA-tagged hStbd1 (hStbd1-HA) and different truncation mutants thereof (ΔN24-HA and ΔC96-HA) for mammalian expression were made by PCR amplification of a human cDNA with addition of an HA tag at the C-terminus. The products were subcloned into BamHI/EcoRI sites of the pcDNA3 vector. Plasmids with double or single point mutations as well as deletion of the potential AIM regions ((W203A, V206A)-HA, (W212A, V215A)-HA, W203A-HA, V206L-HA and Δ198–222–HA) were
Results
The goal of the current study was to identify whether Stbd1 contained a functional AIM motif, which would both define its interacting region with GABARAPL1 and strengthen the argument that Stbd1 shares the basic mechanism for autophagic targeting defined for other Atg8-family interacting cargo receptors. AIM sequences were initially defined by the simple consensus motif WxxL [25] that was later refined to x−3x−2x−1W/F/Yx1x2L/I/V, where x1 and/or x2 and at least one of x−3–x−2–x−1 would be
Discussion
A relationship between glycogen and autophagy or an autophagy-like process has long been inferred even though the mechanism is not well-defined. The fact that Pompe disease is associated with massive over-accumulation of glycogen had implicated a lysosomal degradative pathway [8] and the more recent work of Raben and Plotz has explicitly connected autophagy and glycogen [26]. Physiologic contexts for this process have been described in the liver, muscle and heart of newborn animals [27], [28]
Acknowledgment
Supported in part by NIH grants R37 DK27221 and R01 NS056454.
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Present address: Department of Pharmacology, University of Texas Southwestern, Medical Center, Dallas, TX 75390, United States.