Premium
Cellular Fractionation Identifies the N‐ and C‐ Termini Contributions Toward Membrane Association of the Rho GTPase TCL
Author(s) -
Erickson Jeffery A.,
Esoe Dave-Preston I.,
Tader Brooke R.,
Hamann Michael J.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.06908
Subject(s) - subcellular localization , gtpase , cell fractionation , microbiology and biotechnology , n terminus , biology , gtp' , guanine nucleotide exchange factor , small gtpase , peptide sequence , chemistry , biochemistry , membrane , gene , signal transduction , cytoplasm , enzyme
TCL is a poorly characterized Rho‐family GTPase; however, our lab has performed detailed structure/function analysis of TCL to gain insight into its cellular biochemistry. We have found that its unique N‐terminal extension promotes GTP‐loading of TCL and plasma membrane localization. Deletion of this TCL‐specific sequence promotes localization of TCL to intracellular vesicles. To further explore the role of the N‐terminal and C‐terminal sequences on TCL localization, plasmids encoding Venus were manipulated to include amino acids 1–24 of TCL at the N‐terminus of Venus and amino acids 198–214 at the C‐terminus of Venus. Additional N‐ and C‐terminus deletion mutants of YFP‐tagged TCL were also produced. These plasmids have been transfected into HeLa cells, and subcellular localization of the expressed proteins assessed by fluorescence microscopy and a fractionation procedure. Our results show a clear role for the C‐ terminus of TCL in facilitating membrane localization, but no significant localization directly related to the N‐terminus. Defining how TCL localizes to different cellular membrane environments will help us to understand more clearly its role focal adhesion dynamics, as well as how these dynamics may relate to melanoma development and tumor‐associated angiogenesis. Support or Funding Information This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).