Photosynthesis Research Protocols

METHODS IN MOLECULAR BIOLOGY TM Volume 274 Photosynthesis Research Protocols Edited by Robert Carpentier Grana and Stroma Thylakoids 1 1 Fractionation of Thylakoid Membranes Into Grana and Stroma Thylakoids Juan Cuello and María José Quiles Summary The chloroplasts contain an extensive system of internal membranes or thylakoids in which all the light-harvesting and energy-transducing processes of the photosynthesis are located. Thylakoids are differentiated into stacked membrane regions (or grana thylakoids) and nonstacked membranes (or stroma thylakoids), each with a specialized structure and function. Both kinds of thylakoids can be separated by detergent-based methods or mechanical fragmentation such as sonication. We describe the fractionation of thylakoid membranes into grana and stroma thylakoids by treatment with the detergent digitonin and successive ultracentrifugation of the resultant vesicles. After their separation, the thylakoid fractions retain electron transport and enzymatic activities and are characterized using various parameters. The stroma thylakoids have higher chlorophyll a/chlorophyll b and protein/total chlorophyll ratios, and greater photosystem I and NADH dehydrogenase activities than the grana thylakoids. In the conditions used and on a protein basis of total thylakoids, the yield of stroma thylakoids is 5%, which is considerable taking into account that the stroma thylakoids are a minor component of total thylakoids. Key Words: Barley; chloroplast; digitonin fractionation; grana thylakoids; Hordeum vulgare; stroma thylakoids; thylakoid fractionation. 1. Introduction In photosynthetic eukaryotes, photosynthesis occurs in subcellular organelles known as chloroplasts. These are semiautonomous organelles comprising an envelope formed of two membranes, an aqueous matrix known as stroma, and an extensive system of internal membranes known as thylakoids. All of the light-harvesting and energy-transducing functions are located in the thylakoids, which form a physically continuous membrane system that encloses an aqueous compartment, the thylakoid lumen. With few exceptions, thylakoids are essentially differentiated into stacked membrane regions (grana thylakoids) From: Methods in Molecular Biology, Vol. 274: Photosynthesis Research Protocols Edited by: R. Carpentier © Humana Press Inc., Totowa, NJ 1 2 Cuello and Quiles and non-stacked membranes or membranes exposed to the stroma (stroma thylakoids) (1). Each of these two kinds of thylakoids has a specialized structure and function. Cyclic electron transport occurs in the stroma thylakoids, which account for about 20% of the thylakoid membranes, whereas linear electron transport occurs in the grana thylakoids (2). A quantitative model of the distribution of the photosynthetic components in stroma and grana thylakoids, including the three membrane domains constituting the grana thylakoids, has been proposed (3). The first subfractionations of the thylakoid membranes used detergent-based methods, such as digitonin and Triton X-100 (4). Other methods relied on mechanical fragmentation, such as the passage of thylakoids through a French pressure cell or subjection to sonication (4). More recently, a combination of press treatment or sonication and partitioning in an aqueous polymer two-phase system or countercurrent distribution of vesicles originating from thylakoids has been exploited to isolate subthylakoid fractions (5,6). When necessary, ultrasonic disintegration of the thylakoid membranes into the grana and stroma thylakoid fractions can be used because it prevents possible partial delipidation or the enzymatic inactivations which occur when detergent-based methods are used. Digitonin has proven to be the most useful detergent for isolating vesicles of stroma thylakoids, and lo