A pull-down procedure for the identification of unknown GEFs for small GTPases

TY - JOUR

T1 - A pull-down procedure for the identification of unknown GEFs for small GTPases

AU - Koch, Daniel

AU - Rai, Amrita

AU - Ali, Imtiaz

AU - Bleimling, Nathalie

AU - Friese, Timon

AU - Brockmeyer, Andreas

AU - Janning, Petra

AU - Goud, Bruno

AU - Itzen, Aymelt

AU - Goody, Roger S

PY - 2016/4/2

Y1 - 2016/4/2

N2 - Members of the family of small GTPases regulate a variety of important cellular functions. In order to accomplish this, tight temporal and spatial regulation is absolutely necessary. The two most important factors for this regulation are GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), the latter being responsible for the activation of the GTPase downstream pathways at the correct location and time. Although a large number of exchange factors have been identified, it is likely that a similarly large number remains unidentified. We have therefore developed a procedure to specifically enrich GEF proteins from biological samples making use of the high affinity binding of GEFs to nucleotide-free GTPases. In order to verify the results of these pull-down experiments, we have additionally developed two simple validation procedures: An in vitro transcription/translation system coupled with a GEF activity assay and a yeast two-hybrid screen for detection of GEFs. Although the procedures were established and tested using the Rab protein Sec4, the similar basic principle of action of all nucleotide exchange factors will allow the method to be used for identification of unknown GEFs of small GTPases in general.

AB - Members of the family of small GTPases regulate a variety of important cellular functions. In order to accomplish this, tight temporal and spatial regulation is absolutely necessary. The two most important factors for this regulation are GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), the latter being responsible for the activation of the GTPase downstream pathways at the correct location and time. Although a large number of exchange factors have been identified, it is likely that a similarly large number remains unidentified. We have therefore developed a procedure to specifically enrich GEF proteins from biological samples making use of the high affinity binding of GEFs to nucleotide-free GTPases. In order to verify the results of these pull-down experiments, we have additionally developed two simple validation procedures: An in vitro transcription/translation system coupled with a GEF activity assay and a yeast two-hybrid screen for detection of GEFs. Although the procedures were established and tested using the Rab protein Sec4, the similar basic principle of action of all nucleotide exchange factors will allow the method to be used for identification of unknown GEFs of small GTPases in general.

KW - Guanine Nucleotide Exchange Factors

KW - Saccharomyces cerevisiae

KW - Saccharomyces cerevisiae Proteins

KW - Two-Hybrid System Techniques

KW - rab GTP-Binding Proteins

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1080/21541248.2016.1156803

DO - 10.1080/21541248.2016.1156803

M3 - SCORING: Journal article

C2 - 26918858

VL - 7

SP - 93

EP - 106

JO - Small GTPases

JF - Small GTPases

SN - 2154-1248

IS - 2

ER -