Central Region of Wheat UCP Acts as a Major Signal for Protein Transport to Mitochondria, but Both Distal Regions can also Drive a Protein Import to Mitochondria

Abstract

To identify the region related to the protein transport of mitochondrial uncoupling protein (UCP), we divided wheat UCP (WhUCP1b) protein into three regions (D1, D2 and D3) and fused them with green fluorescent protein (GFP) as an N-terminal signal peptide. Four fusion proteins (WhUCP::GFP, D#::GFP) were expressed in yeast cells to observe the transport of the fusion proteins to mitochondria in vivo. The mitochondrial localization of the four fusion proteins was visualized using GFP fluorescence as a reporter. Western blot analysis also revealed that all four fusion proteins were transported to the mitochondria. The D2 region had the highest protein transport efficiency, and the D1 and D3 regions also had some protein transport activity. Another kind of fusion protein constructed using the D2 region as an internal signal peptide (DSR::D2::GFP) was also successfully targeted to mitochondria. These results demonstrate that the D2 region of WhUCP1b possesses the highest activity directing protein transport to the mitochondria among three regions, and that the protein transport activity of the D2 region is independent on its position within the fusion protein.