Autophagy, a pathway primarily relevant for cell survival, and apoptosis, a process invariably leading to cell death, are the two main mechanisms of cellular self-destruction, which are essential in cell growth, neurodegeneration, tumor suppression, stress and immune response. Currently, a potential crosstalk between apoptosis and autophagy is subject to intensive investigations since recently some direct junctions became obvious. The respective protein-protein interaction network, however, remains to be elucidated in detail. The γ-aminobutyric acid type A (GABA A ) receptor-associated protein GABARAP belongs to a family of proteins implicated in intracellular transport events and was shown to be associated to autophagic processes. Using a phage display screening against the target protein GABARAP, we identified the proapoptotic protein Nix/Bnip3L to be a potential GABARAP ligand. In vitro binding studies, pull-down analysis, coimmunoprecipitation assays and colocalization studies confirmed a direct interaction of both proteins in mammalian cells.

The control of neurotransmitter receptor expression and delivery to the postsynaptic membrane is of critical importance for neural signal transduction at synapses. The sorting, targeting and degradation of neurotransmitter receptors require mechanisms to regulate intracellular vesicular protein transport. These dynamic processes play a key role in the construction and functional maintenance of synapses, and are one of the underlying mechanisms of synaptic plasticity. 4-Aminobutyrate type A (GABA A ) receptor-associated protein (GABA-RAP) is a ubiquitin-like modifier implicated in the intracellular trafficking of GABA A receptors, and belongs to a family of proteins involved in intracellular vesicular transport processes, such as autophagy and intra-Golgi transport. In this article, it is demonstrated that calreticulin is a high affinity ligand of GABARAP. Calreticulin, although best known for its functions as a Ca 2+-dependent chaperone and a Ca 2+ -buffering protein in the endoplasmic reticulum, is also localized to the cytosol and exerts a variety of extra-endoplasmic reticulum functions. By phage display screening of a randomized peptide library, peptides that specifically bind GABARAP were identified. Their amino acid sequences allowed us to identify calreticulin as a potential GABARAP binding protein. GABARAP binding to calreticulin was confirmed by pull-down experiments with brain lysate and colocalization studies in N2a cells. Calreticulin and GABARAP interact with a dissociation constant K d ¼ 64 nm and a mean lifetime of the complex of 20 min. Thus, the interaction between GABARAP and calreticulin is the strongest so far reported for each protein.Abbreviations CaN, calcineurin; CNX, calnexin; CRT, calreticulin; DDX47, DEAD box polypeptide 47; ER, endoplasmic reticulum; GABA A receptor, 4-aminobutyrate type A receptor; GABARAP, GABA A receptor-associated protein; GRIP1, glutamate receptor-interacting protein 1; GST, glutathione S-transferase; Ins(1,4,5)P 3 , inositol 1,4,5-triphosphate; HSQC, heteronuclear single quantum coherence; LC3, light chain 3; MAP1 LC3, microtubule-associated protein 1 light chain 3; N2a, NEURO-2a; NHS, N-hydroxysuccinimide; NSF, N-ethylmaleimide sensitive factor; NaCl ⁄ P i , phosphate buffer pH 7.6; PRIP-1, phospholipase C-related inactive protein type 1; PSSM, position-specific scoring matrix; SPR, surface plasmon resonance; SUMO, small ubiquitin-like modifier; UBL, ubiquitin-like modifier; Ubq, ubiquitin; ULK1, unc-51-like kinase 1.

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