Artificial tethering of LC3 or p62 to organelles is not sufficient to trigger autophagy

• Published in: Cell death & disease 2019-10, Vol.10 (10), p.771-10, Article 771
• Main Authors: Loos, Friedemann, Xie, Wei, Sica, Valentina, Bravo-San Pedro, José Manuel, Souquère, Sylvie, Pierron, Gérard, Lachkar, Sylvie, Sauvat, Allan, Petrazzuolo, Adriana, Jimenez, Ana Joaquina, Perez, Franck, Maiuri, Maria Chiara, Kepp, Oliver, Kroemer, Guido
• Format: Article
• Language: English
• Subjects: 14/19; 14/28; 14/35; 38/109; 631/80/39/2346; 631/92/96; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Antibodies; Autophagy; Autophagy - drug effects; Autophagy - genetics; Avidin; Biochemistry; Biomedical and Life Sciences; Biotin; Biotin - metabolism; Cell Biology; Cell Culture; Cell Line, Tumor; Cellular Biology; Cytosol; Cytosol - metabolism; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Golgi apparatus; Golgi Apparatus - metabolism; Green fluorescent protein; Green Fluorescent Proteins - metabolism; Humans; Immunology; Life Sciences; Microtubule-associated proteins; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - metabolism; Organelles; Phagocytosis; Protein Binding - genetics; Protein Binding - physiology; Protein Transport - genetics; Protein Transport - physiology; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Streptavidin; Streptavidin - metabolism; Subcellular Processes
• ISSN: 2041-4889; 2041-4889
• DOI: 10.1038/s41419-019-2011-5

The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules (“hooks”) targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP–GFP–LC3 and p62–SBP–GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP–GFP–LC3 and p62–SBP–GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.