QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00801

This Article Figures Only Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Galperin, E. Articles by Sorkin, A. Search for Related Content PubMed PubMed Citation Articles by Galperin, E. Articles by Sorkin, A.

Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma-membrane-targeted Rab5 on clathrin-dependent endocytosis

Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262, USA

^* Author for correspondence (e-mail: alexander.sorkin{at}uchsc.edu)

Accepted 25 July 2003

Rab5 is a small GTPase that controls endocytosis and early endosome dynamics. To visualize active, GTP-loaded Rab5 in living cells, we developed molecular sensors consisting of the Rab5-binding fragments of Rabaptin5 or EEA.1 fused to yellow fluorescent protein (YFP). Interaction of these sensors with GTP-bound Rab5 fused to cyan fluorescent protein (CFP) resulted in fluorescence resonance energy transfer (FRET) between CFP and YFP. Activated Rab5 was detected by FRET microscopy in endosomal compartments and often concentrated in microdomains in the endosomal membrane. Although the plasma membrane-localized activity of Rab5 was not detected by light microscopy, overexpression of a GDP-bound mutant of CFP-Rab5(S34N) inhibited internalization of the epidermal growth factor receptor by retaining receptors in clathrin-coated pits. To test whether the Rab5(S34N) mutant affects endocytosis directly at the plasma membrane, CFP-Rab5 was fused to the plasma membrane targeting sequence of K-Ras containing a CAAX motif. The resulting chimeric CFP-Rab5-CAAX was located mainly in the plasma membrane and was capable of binding GTP as judged by FRET microscopy with the Rabaptin5-based sensor. Interestingly, EEA.1 sensor did not follow activated Rab5-CAAX to the plasma membrane, suggesting that the interaction of EEA.1 with Rab5 plays a secondary role in EEA.1 targeting. Overexpression of CFP-Rab5(S34N)CAAX prevented endocytosis of receptors by retaining them in coated pits. These data suggest that the dominant-negative effect of the Rab5(S34N) mutant on the late stages of endocytosis can be mediated through the inhibition of cytosol-associated or plasma-membrane-associated rather than endosome-associated regulators of Rab proteins.

Key words: Rab5, Epidermal Growth Factor, EGF, Endocytosis

This article has been cited by other articles:

				V. A. Torres, A. Mielgo, D. Barila, D. H. Anderson, and D. Stupack Caspase 8 Promotes Peripheral Localization and Activation of Rab5 J. Biol. Chem., December 26, 2008; 283(52): 36280 - 36289. [Abstract] [Full Text] [PDF]

				I. J. Lodhi, D. Bridges, S.-H. Chiang, Y. Zhang, A. Cheng, L. M. Geletka, L. S. Weisman, and A. R. Saltiel Insulin Stimulates Phosphatidylinositol 3-Phosphate Production via the Activation of Rab5 Mol. Biol. Cell, July 1, 2008; 19(7): 2718 - 2728. [Abstract] [Full Text] [PDF]

				P. S. David, R. Tanveer, and J. D. Port FRET-detectable interactions between the ARE binding proteins, HuR and p37AUF1 RNA, September 1, 2007; 13(9): 1453 - 1468. [Abstract] [Full Text] [PDF]

				G. A. Mardones, P. V. Burgos, D. A. Brooks, E. Parkinson-Lawrence, R. Mattera, and J. S. Bonifacino The Trans-Golgi Network Accessory Protein p56 Promotes Long-Range Movement of GGA/Clathrin-containing Transport Carriers and Lysosomal Enzyme Sorting Mol. Biol. Cell, September 1, 2007; 18(9): 3486 - 3501. [Abstract] [Full Text] [PDF]

				M. Shmuel, E. Nodel-Berner, T. Hyman, A. Rouvinski, and Y. Altschuler Caveolin 2 Regulates Endocytosis and Trafficking of the M1 Muscarinic Receptor in MDCK Epithelial Cells Mol. Biol. Cell, May 1, 2007; 18(5): 1570 - 1585. [Abstract] [Full Text] [PDF]

				J. Kalesnikoff, E. J. Rios, C.-C. Chen, S. Nakae, B. A. Zabel, E. C. Butcher, M. Tsai, S.-Y. Tam, and S. J. Galli RabGEF1 regulates stem cell factor/c-Kit-mediated signaling events and biological responses in mast cells. PNAS, February 21, 2006; 103(8): 2659 - 2664. [Abstract] [Full Text] [PDF]

				E. Russinova, J.-W. Borst, M. Kwaaitaal, A. Cano-Delgado, Y. Yin, J. Chory, and S. C. de Vries Heterodimerization and Endocytosis of Arabidopsis Brassinosteroid Receptors BRI1 and AtSERK3 (BAK1) PLANT CELL, December 1, 2004; 16(12): 3216 - 3229. [Abstract] [Full Text] [PDF]

				F. Huang, A. Khvorova, W. Marshall, and A. Sorkin Analysis of Clathrin-mediated Endocytosis of Epidermal Growth Factor Receptor by RNA Interference J. Biol. Chem., April 16, 2004; 279(16): 16657 - 16661. [Abstract] [Full Text] [PDF]