Abstract
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.
Highlights
Macroautophagy is the sole mechanism allowing for the turnover of entire organelles and large protein aggregates, having a major role in cellular adaptation to stress and changing conditions, as well as in the avoidance of premature aging of cytoplasmic components
Based on these physicochemical properties, we built a two-component retention using selective hooks (RUSH) system[21], in which streptavidin is located to different subcellular compartments by fusing it with CD74 or Golgin[84] (Fig. 1a)
We have shown that streptavidin-binding peptide (SBP)–green fluorescent protein (GFP)–LC3 or p62–SBP–GFP behave like endogenous LC3 or p62 upon autophagy induction by rapamycin, the first distributing to cytoplasmic puncta[25], the second reducing its abundance[27], one might argue that these fusion proteins might have changed their native conformation, losing the capacity to interact among each other via the LC3-interacting region (LIR) in p629
Summary
Macroautophagy (hitherto called “autophagy”) is the sole mechanism allowing for the turnover of entire organelles and large protein aggregates, having a major role in cellular adaptation to stress and changing conditions, as well as in the avoidance of premature aging of cytoplasmic components. The regulation of autophagy is complex involving the coordinated activation of protein kinases (in particular the ULK1 kinase complex), lipid kinases (in particular the Beclin 1 complex) and a ubiquitin-like conjugation system (organized around ATG5 and ATG7)[5,6] This latter system assures the C-terminal lipidation (by the attachment of a phosphatidyl ethanolamine group) of proteolytically matured proteins from the microtubule-associated proteins 1 A/1B light chain 3B (hereafter referred to as LC3) family (such as LC3A, LC3B, or GABARAP), increasing their lipophilicity and allowing them to insert into the membrane of nascent phagophores (that engulf autophagic cargo), autophagosomes (that have closed to sequester the cargo) and autolysosomes (that arise from the fusion of autophagosomes with lysosomes)[7,8]. Many assays designed to quantify autophagy monitor the lipidation of LC3 (which increases its electrophoretic mobility) and the subcellular distribution of LC3 toward cytoplasmic “puncta”[9].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
