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Planta : Volume 201, Issue 4, April 1997

Planta : Volume 201, Issue 3, March 1997

Planta : Volume 201, Issue 1, March 1997

Immunogold labelling of cryosectioned pea chloroplasts and initial localization of the proteins associated with the protein import machinery

Content Provider	Springer Nature Link
Author	Morin, Xenia K. Soll, Jürge
Copyright Year	1997
Abstract	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 buffered glutaraldehyde solution and then infiltrated with a buffered solution of 10% polyvinylpyrrolidone in 2.07 M sucrose prior to freezing in liquid nitrogen and sectioning in an ultracryomicrotome. Sections were thawed, immunolabelled, and stained with ammonium molybdate in methyl cellulose on Formvar/carbon-coated Cu or Cu/Pd electron-microscope grids. Cryosectioning gave excellent structural preservation and retained 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-bisphosphate carboxylase/oxygenase (Rubisco), a stromal protein, (ii) the chloroplast ATP synthase (CF1), a peripheral thylakoid protein, and (iii) different envelope membrane proteins. Antibodies raised against three members of the chloroplasticouterenvelopeprotein (OEP) import machinery, 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 localization of OEP86, OEP75 and OEP34 to the outer envelope by biochemical methods was confirmed by our immuno electronmicroscopic analysis. Additionally, 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 intact chloroplasts provides a method for studying the localization of chloroplast proteins, especially those residing in the inner and outer envelope membranes.
Starting Page	119
Ending Page	127
Page Count	9
File Format	PDF
ISSN	00320935
Journal	Planta
Volume Number	201
Issue Number	2
e-ISSN	14322048
Language	English
Publisher	Springer-Verlag
Publisher Date	1997-01-01
Publisher Place	Berlin/Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Chloroplast Envelope proteins Pisum Protein import Tokuyasu technique Agriculture Ecology Forestry Plant Sciences
Content Type	Text
Resource Type	Article
Subject	Genetics Plant Science

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