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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. We employed live cell imaging with particular emphasis on a GABARAP split-tandem construct, GABARAP(G116A)-mTagBFP2-GABARAP (G-B-G), which retains both a free N-terminus and a lipidation-competent c-terminus, while bivalence creates a gain in affinity conferred by avidity. Notably, reminiscent of early in vitro studies demonstrating an interaction of GABARAP and tubulin, our results revealed a robust association of G-B-G with the microtubule network in living cells. We show that the presence of several basic residues in the amino-terminal helical subdomain of GABARAP and avidity emerged as essential for robust MT association, whereas lipidation ability was not decisive. Interestingly, while the position of the FP-tag had little influence on the result, the nature of the FP itself was crucial, with mTagBFP2 being required for tracking GABARAP tandems in the vicinity of MTs. Though artificial effects cannot be excluded, we assume that G-B-G, with its increased avidity, can give visibility to processes that are based on inherently weak interactions, and thus can help elucidate potential roles of GABARAP e.g. in microtubule-associated processes that are integral to autophagy-related and -unrelated cellular transport.
Pubmed ID: 40395516
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Biomedical technology research center and training resource for the study of the structure of partially ordered biological molecules, complexes of biomolecules and cellular structures under conditions similar to those present in living cells and tissues. The goal of research at BioCAT is to determine the detailed structure and mechanism of action of biological systems at the molecular level. The techniques used are X-ray fiber diffraction, X-ray solution scattering and X-ray micro-emission and micro-absorption spectroscopy, with an emphasis on time-resolved studies and the development of novel techniques.
View all literature mentionsNon-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
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