An Improved Intracellular Synthetic Lipidation-Induced Plasma Membrane Anchoring System for SNAP-Tag Fusion Proteins

• Published in: Biochemistry (Easton) Vol. 59; no. 33; pp. 3044 - 3050
• Main Authors: Yoshii, Tatsuyuki, Tahara, Kai, Suzuki, Sachio, Hatano, Yuka, Kuwata, Keiko, Tsukiji, Shinya
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 25.08.2020
• Subjects: Cell Membrane - chemistry; Cell Membrane - metabolism; Escherichia coli; Lipid-Linked Proteins - chemical synthesis; Lipid-Linked Proteins - chemistry; Lipid-Linked Proteins - metabolism; Membrane Lipids - chemistry; Membrane Lipids - metabolism; Protein Engineering - methods; Recombinant Fusion Proteins - chemical synthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Staining and Labeling - methods; Tetrahydrofolate Dehydrogenase - chemistry; Tetrahydrofolate Dehydrogenase - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.0c00410

The ability to chemically introduce lipid modifications to specific intracellular protein targets would enable the conditional control of protein localization and activity in living cells. We recently developed a chemical–genetic approach in which an engineered SNAP-tag fusion protein can be rapidly relocated and anchored from the cytoplasm to the plasma membrane (PM) upon post-translational covalent lipopeptide conjugation in cells. However, the first-generation system achieved only low to moderate protein anchoring (recruiting) efficiencies and lacked wide applicability. Herein, we describe the rational design of an improved system for intracellular synthetic lipidation-induced PM anchoring of SNAP-tag fusion proteins. In the new system, the SNAPf protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAPΔ, is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl-DCys lipopeptidomimetic, called mDcBCP, is used as a cell-permeable chemical probe for intracellular SNAP-tag lipidation. The use of this combination allows significantly improved conditional PM anchoring of SNAP-tag fusion proteins. This second-generation system was applied to activate various signaling proteins, including Tiam1, cRaf, PI3K, and Sos, upon synthetic lipidation-induced PM anchoring/recruitment, offering a new and useful research tool in chemical biology and synthetic biology.