Sequence determinants of a microtubule tip localization signal (MtLS).
Microtubule plus-end-tracking proteins (+TIPs) specifically localize to the growing plus-ends of microtubules to regulate microtubule dynamics and functions. A large group of +TIPs contain a short linear motif, SXIP, which is essential for them to bind to end-binding proteins (EBs) and target microtubule ends. The SXIP sequence site thus acts as a widespread microtubule tip localization signal (MtLS). Here we have analyzed the sequence-function relationship of a canonical MtLS. Using synthetic peptide arrays on membrane supports, we identified the residue preferences at each amino acid position of the SXIP motif and its surrounding sequence with respect to EB binding. We further developed an assay based on fluorescence polarization to assess the mechanism of the EB-SXIP interaction and to correlate EB binding and microtubule tip tracking of MtLS sequences from different +TIPs. Finally, we investigated the role of phosphorylation in regulating the EB-SXIP interaction. Together, our results define the sequence determinants of a canonical MtLS and provide the experimental data for bioinformatics approaches to carry out genome-wide predictions of novel +TIPs in multiple organisms.
Laboratory of Biomolecular Research, Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.
Rubén M
RM
Buey
Indrani
I
Sen
Oliver
O
Kortt
Renu
R
Mohan
David
D
Gfeller
Dmitry
D
Veprintsev
Ines
I
Kretzschmar
Jörg
J
Scheuermann
Dario
D
Neri
Vincent
V
Zoete
Olivier
O
Michielin
José María
JM
de Pereda
Anna
A
Akhmanova
Rudolf
R
Volkmer
Michel O
MO
Steinmetz
D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008868:Microtubule Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D040081:Protein Array Analysis; D011485:Protein Binding; D021382:Protein Sorting Signals / Q000502:physiology*; D012568:Schizosaccharomyces / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029681:Arabidopsis Proteins; D008868:Microtubule Proteins; D021382:Protein Sorting Signals; D029702:Schizosaccharomyces pombe Proteins