IMMUNOGOLD LABELING OF CRYOSECTIONED PEA-CHLOROPLASTS AND INITIAL LOCALIZATION OF THE PROTEINS ASSOCIATED WITH THE PROTEIN IMPORT MACHINERY

The electron-microscopic technique for immunogold labelling of thawed cryosectioned material (K.T. Tokuyasu, 1989, Histochem J 21: 163-171) has been adapted for use with isolated chloroplasts. Percoll-purified pea (Pisum Sativum L. cv Feltham First) chloroplasts were fixed in a b uffered glutaraldehyde solution and then infiltrated with a buffered s olution of 10% polyvinylpyrrolidone in 2.07 M sucrose prior to freezin g in liquid nitrogen and sectioning in an ultracryomicrotome. Sections were thawed, immunolabelled, and stained with ammonium molybdate in m ethyl cellulose on Formvar/carbon-coated Cu or Cu/Pd electron-microsco pe grids. Cryosectioning gave excellent structural preservation and re tained antigenicity, The effectiveness of this technique in localizing proteins to their specific chloroplast compartment was assayed using antibodies raised against: (i) the large subunit of ribulose-1,5-bisph osphate carboxylase/oxygenase (Rubisco), a stromal protein, (ii) the c hloroplast ATP synthase (CF1): a peripheral thylakoid protein, and (ii i) different envelope membrane proteins. Antibodies raised against thr ee members of the chloroplastic outer envelope protein (OEP) import ma chinery, a 34-kDa protein (OEP34 or IAP34), the channel forming 75-kDa protein (OEP75 or IAP75), and the 86-kDa precursor protein receptor ( OEP86 or IAP86) were tested for their localization. The previous local ization of OEP86, OEP75 and OEP34 to the outer envelope by biochemical methods was confirmed by our immune electron-microscopic analysis. Ad ditionally, a constituent of the chloroplastic inner envelope protein (IEP) import machinery IEP 110 (IAP 100) was clearly localized to this membrane. Therefore, cryosectioning and immunogold labelling of intac t chloroplasts provides a method for studying the localization of chlo roplast proteins, especially those residing in the inner and outer env elope membranes.