title: Identification of rab20 as a potential regulator of connexin 43 trafficking.
abstract: Connexin oligomerization and trafficking are regulated processes. To identify proteins that control connexin 43 (Cx43), a screen was designed using HeLa cells expressing a Cx43 construct with di-lysine endoplasmic reticulum (ER)-retention/retrieval motif, Cx43-HKKSL. At moderate levels of expression, Cx43-HKKSL is retained in the ER as monomers; however, Cx43-HKKSL stably overexpressed by HeLa cells localizes to the perinuclear region and oligomerizes. HeLa/Cx43-HKKSL overexpressors were transiently transfected with pooled clones from a human kidney cDNA library and used immunofluorescence microscopy to identify cDNAs that enabled overexpressed Cx43-HKKSL to convert from a perinuclear to ER localization pattern. Using this approach, a small molecular weight GTPase, rab20, was identified as a candidate protein with the ability to regulate Cx43 trafficking. Enhanced green fluorescent protein (EGFP)-tagged rab20 showed a predominantly perinuclear and ER localization pattern and caused wild-type Cx43 to be retained inside the cell. By contrast, mutant EGFP-rab20T19N, which lacks the ability to bind GTP, had no effect on Cx43. These results suggest Cx43 is transported through an intracellular compartment regulated by rab20 along the secretory pathway.
authors.affiliation: Department of Neurology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
authors.forename: Jayasri
authors.initials: J
authors.lastname: Das Sarma
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Kaplan
authors.forename: Dounia
authors.initials: D
authors.lastname: Willemsen
authors.forename: Michael
authors.initials: M
authors.lastname: Koval
mesh_terms: D018031:Connexin 43 / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport / Q000502:physiology; D020691:rab GTP-Binding Proteins / Q000502:physiology*
chemical_list: D018031:Connexin 43; C509832:Rab20 protein, human; D020691:rab GTP-Binding Proteins