Eur. J. Biochem. zyxwvutsrqpon 124, 125- 129 (1982) zyxwvutsr 0 FEBS 1982 zyxwvutsrqpon Processing of a Chloroplast-Translated Membrane Protein zyxw in vivo Analysis of the Rapidly Synthesized 32 000-dalton Shield Protein and Its Precursor in Spivodefa ofigovvhiza Avi REISFELD, Autar K. MATTOO, and Marvin EDELMAN Department of Plant Genetics, The Weizmann Institute zyxwvuts 01' Science, Rehovot (Received October 8/Decetnber 31. 1981) The 32000-dalton (Da) shield protein regulating electron transport in Spirodelu oligorrlrizu is an integral chloroplast membrane polypeptide. It is rapidly synthesized, constituting a major chloroplast-translation product in zyxwvutsrqp vivo. Following in vitro translation of spirodela chloroplast RNA in a wheat germ system, a 33 500-Da polypeptide is produced. Synthesis of a 33500-Da protein, associated with the chloroplast membrane. is also seen in vivo, within 2 min of pulse-labeling spirodela with radioactive amino acids. Comparative analyses among these polypeptides reveal: (a) all three are deficient in lysine residues; (b) the two 33 500-Da species have indistinguishable partial proteolytic digestion patterns while that for the 32000-Da protein differs only slightly from them; (c) radio- activity from the 33 500-Da polypeptide is rapidly chased in vivo into the 32 000-Da protein, even in the presence of protein synthesis inhibitors. These results show the 33500-Da proteins synthesized in vitro and in vivo to be the precursor form of the 32 000-Da shield protein in spirodela, with processing commencing only alter completion of the precursor polypeptide chain and insertion into the membrane. Numerous chloroplast proteins are synthesized as pre- cursor polypeptides, which are subsequently processed to mature forms [I]. Of those investigated, virtually all are synthesized in the cytoplasm and are post-translationally and vectorially transported across the organelle envelope [2,3]. An exception is a rapidly synthesized 32000-Da (32 kDa) protein, recently identified as the shield protein regulating photosystem I1 electron transport in the thylakoids [4] and binding azidoatrazine zyxwvutsrq IS]. This membrane protein, highly conserved among photosynthetic eukaryotes [6], is a chloro- plast gene product [7] and is synthesized within the plastid [8]; yet it has been proposed to be associated with a precursor form [9]. In the present study we describe some properties of the rapidly synthesized 32-kDa polypeptide from the aquatic angiosperm spirodela and detail its relationship to its precursor forms in vivo and in vitro. Preliminary accounts of this work have been published [lo, I I]. EXPERIMENTAL PROCEDURE Isolution of' Total Cell Membranes Axenic Spirodelu oligorrhiza (Kurtz) Hegelm. was cultured phototrophically under steady-state conditions (2000 Ix, cool white fluorescent lights, 25 "C) in half-strength Hutner's medium (cf. [12]). Conditions of labeling with radioactive amino acids (highest spec. act.) are described in legends to the figures. The radiolabeled membrane and supernatant fractions were obtained after machine homogenization at 2 "C (Heidolph 50111, at speed 7) of the tissue by 20 strokes in a conical all-glass homogenizer. Homogenization buffer con- tained 150 mM NaCI, 2.5 mM Tris/glycine, pH 8.6 and 1 mM phenylmethylsulfonyl fluoride (Sigma). The homogenate was centrifuged at 3OOOOxg for 20 min. The pelleted membrane fraction was washed 2 - 3 times with the homogenization buffer and then twice with 2.5 mM Ti-is glycine, pH 8 6, by repeated cycles of resuspension and centrifugation. Finally the washed membrane fraction was su4pended in 2 5 mM Tris/glycine, pH 8.6, and stored at -80 zyx C' Polyucrylamide Gel Electrophoresis Electrophoresis of proteins was carried out on 5odium dodecylsulphate slab gels containing a 10 --20 gradient of polyacrylamide with a 3 "/, stacking gel 215 described [13]. Gels were analyzed by autoradiography or prepared for fluoro- graphy according to Bonner and Lahkey [14], dried and exposed on CURIX RP2 X-ray film (Agfa). Limited pro- teolysis of polypeptides excised from dried gels was carried out on 15 - 20 % dodecylsulphate/pol~acrylamide gradient gels with Staphylococcus aureus V8 protease (Miles) as de- scribed by Cleveland et al. [I51 Other Techniques RNA was prepared from chloroplasts [I61 and translated in a wheat germ cell-free system [17]. [3'S]Methionine-labeled chloroplast membrane and soluble proteins were isolated by the method of Blair and Ellis [18]. Radioactivity incorporated into protein was determined by the method of Mans and Novelli [19]. RESULTS The gel electrophoretic patterns of newly synthesked polypeptides located within the chloroplasts of spirodela are shown in Fig. 1. The major stromal components labeled with [35S]methionine are the large (LS) and mall (SS) subunits of ribulose bisphosphate carboxylase (lane 1). Among the thylakoid membrane polypeptides the 32-kDa polypeplide predominates (lane 2). The association of the 32-kDa protein