A trafficking motif alters GEVI activity implicating persistent protein interactions at the membrane

Sungmoo Lee; Bok Eum Kang; Yoon-Kyu Song; Bradley J Baker

doi:10.1016/j.bpr.2022.100047

A trafficking motif alters GEVI activity implicating persistent protein interactions at the membrane

Biophys Rep (N Y). 2022 Feb 22;2(2):100047. doi: 10.1016/j.bpr.2022.100047. eCollection 2022 Jun 8.

Authors

Sungmoo Lee^{1

2}, Bok Eum Kang¹, Yoon-Kyu Song², Bradley J Baker^{1

3}

Affiliations

¹ Brain Science Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea.
² Graduate School of Convergence Science and Technology, Seoul National University, Seoul, Republic of Korea.
³ Division of Bio-Medical Science and Technology, KIST School, University of Science and Technology (UST), Seoul, Republic of Korea.

Abstract

Efficient plasma-membrane expression is critical for genetically encoded voltage indicators (GEVIs). To improve the plasma-membrane expression, we introduced multiple combinations of plasma-membrane trafficking motifs at different positions to members of the Bongwoori family of GEVIs. An improvement from 20% to 27% in the ΔF/F/100 mV depolarization of the plasma membrane was observed when a Golgi transport motif was inserted near the N-terminus in conjunction with an endoplasmic reticulum release motif near the C-terminus of the protein. Unfortunately, this variant was also slower. The weighted tau on of the variant (25 ms) was more than double the original construct (11 ms). The weighted tau off was >20 ms compared with 10 ms for the original GEVI. The voltage range of the GEVI was also shifted to more negative potentials. Insertion of spacer amino acids between the fluorescent-protein domain and the endoplasmic reticulum release motif at the C-terminus rescued the speed of both the tau on and tau off while restoring the voltage range and maintaining the improved voltage-dependent optical signal. These results suggest that while trafficking motifs do improve plasma-membrane expression, they may also mediate persistent associations that affect the functioning of the protein.