Interactions between EB1 and microtubules: dramatic effect of affinity tags and evidence for cooperative behavior.
Plus end tracking proteins (+TIPs) are a unique group of microtubule binding proteins that dynamically track microtubule (MT) plus ends. EB1 is a highly conserved +TIP with a fundamental role in MT dynamics, but it remains poorly understood in part because reported EB1 activities have differed considerably. One reason for this inconsistency could be the variable presence of affinity tags used for EB1 purification. To address this question and establish the activity of native EB1, we have measured the MT binding and tubulin polymerization activities of untagged EB1 and EB1 fragments and compared them with those of His-tagged EB1 proteins. We found that N-terminal His tags directly influence the interaction between EB1 and MTs, significantly increasing both affinity and activity, and that small amounts of His-tagged proteins act synergistically with larger amounts of untagged proteins. Moreover, the binding ratio between EB1 and tubulin can exceed 1:1, and EB1-MT binding curves do not fit simple binding models. These observations demonstrate that EB1 binding is not limited to the MT seam, and they suggest that EB1 binds cooperatively to MTs. Finally, we found that removal of tubulin C-terminal tails significantly reduces EB1 binding, indicating that EB1-tubulin interactions are mediated in part by the same tubulin acidic tails utilized by other MAPs. These binding relationships are important for helping to elucidate the complex of proteins at the MT tip.
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Zhiqing C
ZC
Zhu
Kamlesh K
KK
Gupta
Aranda R
AR
Slabbekoorn
Benjamin A
BA
Paulson
Eric S
ES
Folker
Holly V
HV
Goodson
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular; D006801:Humans; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013552:Swine; D014404:Tubulin / Q000737:chemistry
C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins; D011108:Polymers; D014404:Tubulin