Determination of the membrane topology of lemur tyrosine kinase 2 (LMTK2) by fluorescence protease protection

Abstract

Lemur tyrosine kinase 2 (LMTK2) is a novel membrane-anchored kinase reported to be involved in several normal and pathophysiological conditions, including endosomal membrane recycling, prostate cancer, and neurodegeneration. In this study, we have investigated the topology and orientation of LMTK2 within cellular membranes utilizing fluorescence protease protection. Appending the green fluorescent protein to either the amino or carboxyl terminus of LMTK2, we were able to determine which side of intracellular membrane these regions were located. Our results indicate that LMTK2 is an integral membrane protein in which both the amino and carboxyl termini are exposed to the cytoplasm. Moreover, this topology places the kinase active site within the cytoplasm.