Fig. 4. PHR1 can form dimers. (A) Yeast two-hybrid PHR1 preys obtained using PHR1a as bait. (B) PHR1 isoforms can form heteromers. Top panel, PHR1 constructs used for the in vitro binding assay. Middle panel, GST-PHR1a or GST alone was incubated with full length [³⁵S]PHR1a, [³⁵S]PHR1b, or [³⁵S]PHR1d. GST-PHR1a interacts with every PHR1 isoform. Lower panel, PHR1.JMD is sufficient for PHR1 homomerisation. (C) Gel filtration analysis of the purified PH domains of PHR1a (PHR1a.PH) and ßV spectrin (ßV spectrin.PH). The figure shows the regression curve between the molecular mass and the partition coefficient (K_av) defined by K_av = (Ve – Vo)/(Vt – Vo), where Ve is elution volume of the protein, Vo, column void volume and Vt, total bed volume. The molecular masses of eluted fractions are estimated according to the fractions corresponding to four molecular mass standards: bovine serum albumin (BSA), ovalbumin, ribonuclease A and chymotrypsinogen A. PHR1a.PH is eluted as a dimer, whereas ßV spectrin.PH is eluted as a monomer. (D) Schematic diagram illustrating how a PHR1 dimer can recruit two myosin 1c, two myosin VIIa, or one myosin 1c and one myosin VIIa molecule to the plasma membrane.