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Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2008) 32, 1318–1322 (Printed in Great Britain)
Interactions between the budding yeast IQGAP homologue Iqg1p and its targets revealed by a split-EGFP bimolecular fluorescence complementation assay
Sevvel Pathmanathan, Emma Barnard and David J. Timson*
School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK


Abstract

A split-EGFP based bimolecular fluorescence complementation (BiFC) assay has been used to detect interactions between the Saccharomyces cerevisiae cytoskeletal scaffolding protein Iqg1p and three targets: myosin essential light chain (Mlc1p), calmodulin (Cmd1p) and the small GTPase Cdc42p. The format of the BiFC assay used ensures that the proteins are expressed at wild type levels thereby avoiding artefacts due to overexpression. This is the first direct in vivo detection of these interactions; in each case, the complex is localised to discrete regions of the yeast cytoplasm. The labelling with EGFP fragments results in changes in growth kinetics, cell size and budding frequency. This is partly due to the reassembled EGFP locking the complexes into essentially permanent interactions. The consequences of this for Iqg1p interactions and BiFC assays in general are discussed.


Key words: Cytoskeleton, Protein-fragment complementation assay (PCA), Calmodulin, Myosin light chain, CDC42, Enhanced green fluorescent protein (EGFP).

*Corresponding author. Tel.: +44 (0)28 9097 5875; fax: +44 (0)28 9097 5877.


Received 14 February 2008; accepted 7 July 2008

doi:10.1016/j.cellbi.2008.07.008


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)