Organelle Targeting of Myosin XI Is Mediated by Two Globular Tail Subdomains with Separate Cargo Binding Sites

Jian-Feng Li,Andreas Nebenführ

doi:10.1074/jbc.m700645200

Abstract

Myosin XI are actin-based molecular motors that are thought to drive organelle movements in plants, analogous to myosin V in animals and fungi. Similar domain structure of these myosins suggests that binding to organelles may occur via the globular tail domain in both types of motors, even though sequence similarity is low. To address this hypothesis, we developed a structure homology model for the globular tail of MYA1, a myosin XI from Arabidopsis, based on the known structure of yeast myosin V (Myo2p) globular tail. This model suggested an interaction between two subdomains of the globular tail which was verified by yeast two-hybrid assay and by in vivo bimolecular fluorescence complementation (BiFC). Interface mapping demonstrated that this subdomain interaction depends critically on the C terminus of helix H6 as well as three specific residues in helices H3 and H15, consistent with the structural prediction. The reconstituted globular tails of several Arabidopsis myosin XIs in BiFC assays targeted to peroxisomes in plant cells, identifying this domain as sufficient for cargo binding. Unlike myosin V, either subdomain of myosin XI alone was targeting-competent and responsible for association with different organelles. In addition, our data suggest that organelle binding is regulated by an allosteric interaction between two tail subdomains. We conclude that the globular tail of myosin XI shares a similar structure with that of myosin V, but has evolved plant-specific cargo binding mechanisms.

Highlights

(6 – 8), or genome-wide sequence analysis [9, 10]
Based on these similarities and the well-studied cargo binding paradigm of myosin V globular tail [23], it is tempting to speculate that the globular tail of myosin XI is responsible for organelle targeting in plant cells
The general structure of the globular tail appeared conserved between myosin V and XI, we found novel mechanisms of cargo binding in myosin XI

Summary

EXPERIMENTAL PROCEDURES

Bioinformatic Analysis—The amino acid sequences of the globular tail of myosin XI (MYA1) and myosin V (Myo2p) were initially aligned by MUSCLE [32] and T-coffee [33] programs. Manual refinement was made by considering the known secondary structure of Myo2p globular tail and the secondary structure of MYA1 globular tail predicted by the Protein Homology Recognition Engine server. Based on this sequence alignment and the Myo2p structure template (PDB code 2F6HX), a homology model of MYA1 globular tail was built by the Swiss-Model server [34]. The resulting PDB file is available online as supplemental data. Plasmid Construction—All PCR primers used and recombinant plasmids constructed in this study are listed in supplemental Tables S1 and S2, respectively, which are available online. For BiFC constructs, the coding sequence for N-terminal 154 resi-

The abbreviations used are

RESULTS

CONCLUSIONS