Identification of rab20 as a potential regulator of connexin 43 trafficking.
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.
Das Sarma
Jayasri
J
Department of Neurology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
Kaplan
Benjamin E
BE
Willemsen
Dounia
D
Koval
Michael
M
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*
D018031:Connexin 43; C509832:Rab20 protein, human; D020691:rab GTP-Binding Proteins