Binding properties of NADPH‐protochlorophyllide oxidoreductase as revealed by detergent and ion treatments of isolated and immobilized prolamellar bodies

Grevby, Cecilia; Engdahl, Sheila; Ryberg, Margareta; Sundqvist, Christer

doi:10.1111/j.1399-3054.1989.tb05382.x

Cited by 38 publications

(25 citation statements)

References 31 publications

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“…13,43,45,46]. Native, membraneassociated pchlide reductase has been shown to partly resist thermolysin treatment [29], salt wash [19,50] and detergents [19], indicating that pchlide reductase is firmly attached to plastid inner membranes in vivo. This suggests that the ability of the pchlide reductase to withstand proteolysis by exogenously added thermolysin can be used to monitor physiological pchlide reductasemembrane association in vitro.…”

Section: Discussionmentioning

confidence: 99%

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts

1995

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The NADPH-protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) is the major protein in the prolamellar bodies (PLBs) of etioplasts, where it catalyzes the light-dependent reduction of protochlorophyllide to chlorophyllide during chlorophyll synthesis in higher plants. The suborganellar location in chloroplasts of light-grown plants is less clear. In vitro assays were performed to characterize the assembly process of the pchlide reductase protein in pea chloroplasts. Import reactions employing radiolabelled precursor protein of the pchlide reductase showed that the protein was efficiently imported into fully matured green chloroplasts of pea. Fractionation assays following an import reaction revealed that imported protein was targeted to the thylakoid membranes. No radiolabelled protein could be detected in the stromal or envelope compartments upon import. Assembly reactions performed in chloroplast lysates showed that maximum amount of radiolabelled protein was associated to the thylakoid membranes in a thermolysin-resistant conformation when the assays were performed in the presence of hydrolyzable ATP and NADPH, but not in the presence of NADH. Furthermore, membrane assembly was optimal at pH 7.5 and at 25 degrees C. However, further treatment of the thylakoids with NaOH after an assembly reaction removed most of the membrane-associated protein. Assembly assays performed with the mature form of the pchlide reductase, lacking the transit peptide, showed that the pre-sequence was not required for membrane assembly. These results indicate that the pchlide reductase is a peripheral protein located on the stromal side of the membrane, and that both the precursor and the mature form of the protein can act as substrates for membrane assembly.

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Section: Discussionmentioning

confidence: 99%

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts

1995

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“…It was found tightly bound to the membrane (Grevby et al 1989;Widell-Wigge and Selstam 1990), even though POR was described as a non-transmembrane protein (Benli et al 1991;Timko 1993;Birve et al 1996). The possibility of POR and its isoforms as being integral membrane proteins invites to new discussions concerning their membrane connection and role in PLB formation as well as their aggregation and interaction with pigments and other proteins, e.g.…”

Section: The Isolation Of Plbs and Ptsmentioning

confidence: 96%

Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

2007

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The prolamellar body (PLB) proteome of dark-grown wheat leaves was characterized. PLBs are formed not only in etioplasts but also in chloroplasts in young developing leaves during the night, yet their function is not fully understood. Highly purified PLBs were prepared from 7-day-old dark-grown leaves and identified by their spectral properties as revealed by low-temperature fluorescence spectroscopy. The PLB preparation had no contamination of extra-plastidal proteins, and only two envelope proteins were found. The PLB proteome was analysed by a combination of 1-D SDS-PAGE and nano-LC FTICR MS. The identification of chlorophyll synthase in the PLB fraction is the first time this enzyme protein was found in extracts of dark-grown plants. This finding is in agreement with its previous localization to PLBs using activity studies. NADPH:protochlorophyllide oxidoreductase A (PORA), which catalyses the reduction of protochlorophyllide to chlorophyllide, dominates the proteome of PLBs. Besides the identification of the PORA protein, the PORB protein was identified for the first time in dark-grown wheat. Altogether 64 unique proteins, representing pigment biosynthesis, photosynthetic light reaction, Calvin cycle proteins, chaperones and protein synthesis, were identified. The in number of proteins' largest group was the one involved in photosynthetic light reactions. This fact strengthens the assumption that the PLB membranes are precursors to the thylakoids and used for the formation of the photosynthetic membranes during greening. The present work is important to enhance our understanding of the significance of PLBs in chloroplast development.

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“…The weak fluorescence around 645 is seen only after Gaussian resolution of the fluorescence spectra (Boddi et al 1992). Thus the Pchlide636-657 can also be designated Pchlide636_645-This pigment is probably associated with the POR but in a non-aggregated state (Grevby et al 1989). It is a minor component of the pigments in the PLBs, but one of the major components of the PTs.…”

Section: Pchlide Spectral Forms and Photoreductionmentioning

confidence: 96%

With chlorophyll pigments from prolamellar bodies to light-harvesting complexes

Sundqvist¹,

Dahlin²

1997

Physiol Plant

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With chlorophyll pigments from prolamellar bodies to light-harvesting complexesChrister Sundqvist and Clas Dahlin Sundqvist, C. and Dahlin, C, 1997. With chlorophyll pigments from prolamellar bodies to light-harvesting complexes. -Physiol. Plant 100: 748-759.The biosynthetic chain leading from 5-aminolevulinic acid to chlorophyll is localised to the plastid. Many of the enzymes are nuclear-encoded. NADPH-protochlorophyllide oxidoreductase (EC 1.3.1.33) is one such enzyme which is encoded by two different genes and can exist in an A and a B form. Its import into the plastid seems to be facilitated when protochlorophyllide is present in the chloroplast envelope. Within the plastid the reductase is assembled to thylakoids or prolamellar bodies. The specific properties of the reductase together with the specific properties of the lipids present in the etioplast inner membranes promote the formation of the three-dimensional regular network of the prolamellar bodies. The reductase forms a temary complex with protochlorophyllide and NADPH that gives rise to different spectral forms of protochlorophyllide. Light transforms protochlorophyllide into chlorophyllide and this photoreaction induces a conformational change in the reductase protein which leads to a process of disaggregation of enzyme, pigment aggregates and membranes, which can be followed spectroscopically and with electron microscopy. The newly formed chlorophyllide is esterified by a membrane-bound nuclear-encoded chlorophyll synthase and the chlorophyll molecule is then associated with proteins into active pigment protein complexes in the photosynthetic machinery.

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Binding properties of NADPH‐protochlorophyllide oxidoreductase as revealed by detergent and ion treatments of isolated and immobilized prolamellar bodies

Cited by 38 publications

References 31 publications

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts

The in vitro assembly of the NADPH-protochlorophyllide oxidoreductase in pea chloroplasts

Proteomic analysis of highly purified prolamellar bodies reveals their significance in chloroplast development

With chlorophyll pigments from prolamellar bodies to light-harvesting complexes

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