Analysis of Organelle Targeting by DIL Domains of the Arabidopsis Myosin XI Family

Abstract

The Arabidopsis thaliana genome encodes 13 myosin XI motor proteins. Previous insertional mutant analysis has implicated substantial redundancy of function of plant myosin XIs in transport of intracellular organelles. Considerable information is available about the interaction of cargo with the myosin XI-homologous yeast myosin V protein myo2p. We identified a region in each of 12 myosin XI sequences that correspond to the yeast myo2p secretory-vesicle binding domain (the “DIL” domain). Structural modeling of the myosin DIL domain region of plant myosin XIs revealed significant similarity to the yeast myo2p and myo4p DIL domains. Transient expression of YFP fusions with the Arabidopsis myosin XI DIL domain resulted in fluorescent labeling of a variety of organelles, including the endoplasmic reticulum, peroxisomes, Golgi, and nuclear envelope. With the exception of the YFP::MYA1 DIL fusion, expression of the DIL–YFP fusions resulted in loss of motility of labeled organelles, consistent with a dominant-negative effect. Certain fusions resulted in localization to the cytoplasm, plasma membrane, or to unidentified vesicles. The same YFP-domain fusion sometimes labeled more than one organelle. Expression of a YFP fusion to a yeast myo2p DIL domain resulted in labeling of plant peroxisomes. Fusions with some of the myosin XI domains resulted in labeling of known cargoes of the particular myosin XI; however, certain myosin XI YFP fusions labeled organelles that had not previously been found to be detectably affected by mutations nor by expression of dominant-negative constructs.

Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells

“…Previously Li and Nebenfuhr (2007) expressed larger regions of the Arabidopsis XI-1 and XI-2 tails that encompass the PAL sub-domain and found they were targeted to peroxisomes, unidentified vesicles, or remained in the cytoplasm (Figure 9). We recently reported that the DIL domain of Arabidopsis myosin XIs, which is C-terminal to the PAL sub-domain and also encompassed by the constructs used by Li and Nebenfuhr (2007), targets YFP to several organelles and membranes (Sattarzadeh et al, 2011). PAL and DIL regions from particular myosin XIs can target YFP to Golgi, nuclear envelope, and/or plasma membrane.…”

“…However, no DIL domains resulted in localization of YFP to mitochondria or tonoplast. Conversely, none of the PAL sub-domain fusions resulted in labeling of ER nor peroxisomes, while 8 DIL domains targeted YFP to peroxisomes and two labeled ER (Sattarzadeh et al, 2011). Thus there are some general differences in the localization information provided by the two different tail regions.…”

“…Constructs diagramed are (1) from Sattarzadeh et al (2011), (2) from Reisen and Hanson (2007), (3) from Li and Nebenfuhr (2007), (4) from Sparkes et al (2008), and (5) from Avisar et al (2009). CC: coiled-coil.…”

“…Regions homologous to the two domains exist in the Arabidopsis myosin XI family. Previously we have described the labeling of subcellular locations by YFP fusions to the DIL (dilute) domain (similar to the yeast secretory vesicle-specific domain) of 12 Arabidopsis myosin XIs (Sattarzadeh et al, 2011). Peroxisomes were labeled by YFP fusions to DIL domains of 8 different myosin XIs, three labeled Golgi, and others labeled the plasma membrane, endoplasmic reticulum (ER), nuclear envelope, and unidentified vesicles, but none interacted with mitochondria (Sattarzadeh et al, 2011).…”

“…Previously we have described the labeling of subcellular locations by YFP fusions to the DIL (dilute) domain (similar to the yeast secretory vesicle-specific domain) of 12 Arabidopsis myosin XIs (Sattarzadeh et al, 2011). Peroxisomes were labeled by YFP fusions to DIL domains of 8 different myosin XIs, three labeled Golgi, and others labeled the plasma membrane, endoplasmic reticulum (ER), nuclear envelope, and unidentified vesicles, but none interacted with mitochondria (Sattarzadeh et al, 2011). Inspection of the members of the Arabidopsis myosin XI gene family revealed that except for XI-J, Arabidopsis myosin XI genes encode a 42 amino-acid region within the tail region that is homologous to the yeast myo2p region known to affect vacuole and mitochondrial inheritance (Figure 1).…”