TCL/RhoJ Vesicular Localization is Regulated by GDP‐loading of its Core GTPase Domain

Abstract

TCL (RhoJ) belongs to the Rho family of GTPases, and while it has been shown to contribute to angiogenesis and metastatic melanoma, less is known about its molecular and cellular function. To better understand the inherent cellular biochemistry of TCL, our lab has been performing a detailed structure/function analysis of the GTPase. Previously, the extended N terminus of TCL was found to be important for GTP‐loading and localization to the plasma membrane. The deletion or mutation of this TCL‐specific sequence led to inactivation of TCL and its localizing to intracellular vesicles. To determine if the N‐terminus of TCL contributes to cellular localization by itself, expression plasmids were produced where the first 24 N‐terminal amino acids and last 21 C‐terminal amino acids of TCL were fused to the N‐ and C‐termini (respectively) of the fluorescent protein Venus. Interestingly, expression of the constructs bearing only the C‐terminal tail of TCL or both the N‐ and C‐termini sequestered Venus to the plasma membrane, indicating the C‐terminal tail is potentially sufficient for TCL membrane localization. Additionally, experiments using constitutively active (CA) and dominant negative (DN) mutants showed CA TCL localized to the plasma membrane while DN TCL was vesicular; however, CA TCL where C terminus has been deleted results in diffuse cytoplasmic localization, while the same deletions in the context of the DN mutation lead to a marked vesicular localization. Together, our results suggest TCL is specifically retained to vesicles through an interaction with an as yet unidentified binding partner, and future experiments will use biotinligase tags of CA and DN TCL and BioID procedures to determine what hypothetical partner protein is retaining TCL to vesicular membranes.Support or Funding InformationSupport was provided by the Nielson Foundation, Bemidji, MN and a grant from Regenerative Medicine Minnesota (RMM‐2017‐EP‐04)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.