STRUCTURE OF ROUGH, SMOOTH, STRIPPED AND RECONSTITUTED ROUGH MEMBRANES DERIVED FROM RAT LIVER AS VISUALIZED BY THE FREEZE FRACTURE TECHNIQUE ABRAHAM A. HOCHBERG, HENRYKH. CZOSNEK,YEHUD1TREICHLER, ITZHAK OHAD, and NATHANDE GROOT Department o f Biological Chemistry, The Hebrew University o f Jerusalem, Jerusalem, Israel (Received 8 September, 1975) Abstract. The structure of purified fractions of rough, smooth, stripped rough and reconstituted rough membranes have been investigated by the freeze etching technique. Preparations of rough and reconstituted rough membranes, active in protein synthesis, show vesicles whose outer surface is covered with ribosome-like particles. The inner surface of these vesicles contains also numerous particles of the same size. The particles located on the outer surface are largely absent in the stripped rough membrane preparations which, however, retain the particles located on the inner face. Particles were not seen either on the outer nor on the inner face of the smooth membranes. The possibility is considered that the particles located on the inner face are specific to the rough membranes and might play a role in the specific binding of ribosomes to the membranes. I. INTRODUCTION The successful reconstitution of a rough membrane active in protein synthesis from stripped rough membrane and free polyribosomes is considered to be an important step in the under- standing of the mechanism of the synthesis of exportable proteins. Binding of free polyribosomes to stripped rough membrane has been reported by several labora- tories [1-4]. The attachment of free polyribosomes to stripped rough membrane has been, so far, evaluated by comparison with the native rough membrane using the following criteria : RNA/ protein ratio, buoyant density [4], morphology as tested by electron microscopal observation of thin sections [5] and incorporation of amino acids into polypeptides [3]. However, on the basis of these criteria, one cannot distinguish between non-specific binding or absorption of free poly- ribosomes to the membrane, which will result in changes in the buoyant density, RNA/protein ratio, and morphological aspects similar to those expected for a functional reconstituted complex. Even incorporation of amino acids promoted by poly(U) or endogenous mRNA [6] into peptides by the isolated reconstituted rough membrane, cannot be considered as a conclusive proof of the in vitro formation of functional rough membrane, since these activities associated with the free polyribosomes will be preserved after binding or absorption to the membrane if the membrane does not exert an inhibitory effect on the amino acid incorporation activity. An important 311 Molecular Biology Reports 2 (1975) 311-319. All Rights Reserved. Copyright Q 1975 by D. Reidel Publishing Company, Dordreeht-Holland.