Interaction of peroxisomes with microtubules - In vitro studies using a novel peroxisome-microtubule binding assay

The association of membrane-bounded cell organelles to microtubules is cruc ial for determination of their shape, intracellular localization and transl ocation. We have shown previously the high affinity binding of peroxisomes to microtubules which appears to be of static nature as in vivo studies ind icate that only a few peroxisomes move along the microtubular tracks. In or der to characterize the interactions of peroxisomes with microtubules, we h ave developed a semiquantitative in vitro binding assay, which is based on the association of highly purified rat liver peroxisomes to microtubules co ated onto microtiterplates. The binding was visualized by differential inte rference contrast and immunofluorescence using a confocal laser scanning mi croscope. The binding was concentration dependent and saturable, being affe cted by time, temperature, and pH. Addition of ATP or the motor proteins ki nesin and dynein increased the binding capacity, while ATP-depletion or mic rotubule associated proteins (MAPs) decreased it. KCl treatment of peroxiso mes reduced the binding, which was restored by dialyzed KCl-stripping eluat e as well as by rat liver cytosol. The reconstituting effect of cytosol was abolished by its pretreatment with proteases or N-ethylmaleimide. Moreover , the treatment of peroxisomes with proteases or N-ethylmaleimide reduced t heir binding, which was not reversed by cytosol. These results suggest the involvement of a peroxisomal membrane protein and cytosolic factor(s) in th e binding of peroxisomes to microtubules. This notion is supported by the o bservation that distinct subfractions of dialyzed KCl-stripping eluate obta ined by gel chromatography augmented the binding. Those subfractions, as we ll as purified peroxisome fractions, exhibited strong immunoreactivity with an antibody to cytoplasmic linker protein (CLIP)-115, revealing a 70-kDa p olypeptide. Moreover, immunodepletion of KCl-stripping eluate and its subfr actions with an antibody to the conserved microtubule binding domain of CLI Ps, abolished their promoting effect on the binding, thus suggesting the in volvement of a CLIP-related protein in the binding of peroxisomes to microt ubules.