RT Journal Article
SR Electronic
T1 Palmitoylation of the oncogenic RhoGEF TGAT is dispensable for membrane localization and consequent activation of RhoA
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 062729
DO 10.1101/062729
A1 Jakobus van Unen
A1 Dennis Botman
A1 Taofei Yin
A1 Yi I. Wu
A1 Mark A. Hink
A1 Theodorus W.J. Gadella, Jr.
A1 Marten Postma
A1 Joachim Goedhart
YR 2016
UL http://biorxiv.org/content/early/2016/11/01/062729.abstract
AB Rho guanine exchange factors (RhoGEFs) control many aspects of the cellular cytoskeleton, and thereby regulate and control processes such as cell migration, cell adhesion and proliferation. TGAT is a splice variant of the RhoGEF Trio, with oncogenic potential. Whether the subcellular location of TGAT is critical for its activity is unknown. Confocal microscopy of fluorescent protein tagged TGAT revealed co-localization with a Golgi marker. Because plasma membrane localized RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was studied. In order to quantitatively measure plasma membrane association we developed a novel, highly sensitive image analysis method. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGATΔ15, lost membrane association. The functional role of membrane localization was determined by measuring TGAT activity in single cells with a RhoA FRET-sensor and F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGATΔ15 variant. Synthetic recruitment of TGATΔ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. Together, these results show that membrane association of TGAT is critical for its activity, but that palmitoylation is dispensable.