Biochimica et Biophysica Acta 861 (1986) 67-73 67 Elsevier BBA 73278 Effects of phospholipases C and D on ordering of channel proteins in the mitochondrial outer membrane Carmen A. Mannella Wadsworth Center for Laboratories and Research, New York State Department of Health and School of Public Health Sciences, State University of New York at Albany, Albany, NY 12201 (U.S.A) (Received 8 April 1986) Key words: Phospholipase;Channel protein ordering; (Mitochondrialouter membrane) The effects of phospholipases C and D on the state of order of the channels in the outer membranes of Neurospora mitochondria have been investigated by negative-stain electron microscopy and optical diffrac- tion. Unlike the situation with phospholipase A ~, treatment of the isolated membranes with phospholipase C or D does not induce crystallization of the channels in the membrane plane. Furthermore, treatment of already-formed periodic arrays of outer membrane channels with either phospholipase C or D causes loss of long-range order in the arrays. The latter result suggests that zwitterionic phospholipids may play an important role in stabilizing the periodic arrays of the channel-forming protein in this membrane. Introduction The principal protein component of outer membranes of Neurospora mitochondria is the 31 kDa pore-former, called VDAC or mitochondrial porin [1-4]. Treatment of these membranes with low levels of soluble phospholipase A 2 during dialysis against low-salt buffer induces crystalliza- tion of the pore protein in the membrane plane [5,6]. The concomitant decrease in surface area and increase in buoyant density of the mitochon- drial outer membranes suggest that ordering of this integral protein is associated with removal of phospholipids from the membranes, presumably after their hydrolysis to lysophospholipids and free fatty acids by phospholipase A 2 [5,6]. Earlier observations of periodic arrays of the pore protein on untreated outer membranes from Neurospora Correspondence address: Wadsworth Center for Laboratories and Research, New York State Department of Health, Empire State Plaza, Albany, NY 12201 U.S.A. mitochondria [1,4] suggest that endogenous mitochondrial phospholipase A 2 activity [7,8] may be effective in inducing ordering of the channels. Such phospholipase A z-induced crystallization of mitochondrial outer membrane channels may have functional implications. For example, it has re- cently been shown that clustering of acetylcholine receptors alters their channel kinetics [9]. How- ever, at present, our primary interest in this phe- nomenon of phospholipase-induced ordering of membrane proteins is a practical one, i.e., the preparation of large two-dimensional crystalline arrays for low-dose electron microscopy and image analysis [10]. The phospholipase A z-dialysis technique has been successfully applied to the planar crystalliza- tion of at least one other membrane protein, the CaZ+-ATPase of sarcoplasmic reticulum [11]. The relative speed and simplicity of the technique makes it an attractive starting point when attempt- ing to crystallize proteins present in large con- centration in native or reconstituted membranes. In this report, we examine the effects of two 0005-2736/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomodicalDivision)