Highlighting the hidden: A tagging strategy for monitoring the association of GABARAP with microtubules in living cells

GABARAP, like other ATG8 proteins, is a ubiquitin-like modifier and its C-terminal lipid conjugation enables association with cellular membranes. To prevent interference with the lipidation process, N-terminal fluorescent protein (FP) tagging strategies have become the standard for studying ATG8 localization and function in living cells, significantly contributing to our understanding of this protein family’s multifaceted roles. However, recent findings have unveiled potential limitations of bulky N-terminal tags, particularly regarding ATG8 functionality and localization in specific contexts. This study employed live cell imaging with particular emphasis on the GABARAP split-tandem construct, GABARAP(G116A)-mTagBFP2-GABARAP (G- B -G), which retains both a free N-terminus and a lipidation-competent C-terminus. Notably, our results revealed a robust association of G- B -G with the microtubule network in living cells which was not observed with N-terminal FP fusions of GABARAP, although early in vitro studies demonstrated an interaction of GABARAP and tubulin. Since we observed alteration of the microtubule network organization for G- B -G, this construct emerges as a valuable tool, which can help shedding light on potential roles of GABARAP in microtubule-associated processes that are integral to autophagy-related and -unrelated cellular transport. ### Competing Interest Statement The authors have declared no competing interest. * Abbreviations: ATG8 : autophagy related protein 8 FP : fluorescent protein GABARAP : γ-aminobutyric acid type A receptor-associated protein HA-tag : hemagglutinin tag Icorr : Index of correlation KO : knockout LDS : LIR docking site LIR : LC3-interacting region MAP1LC3/LC3 : microtubule associated protein 1 light chain 3 MAP : microtubule associated protein MT : microtubule nMDP : normalized mean deviation product ROI : region of interest SiR : Silicon rhodamine