Comment
doi: 10.1073/pnas.061500998.
VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation
Affiliations
- PMID: 11274447
- PMCID: PMC31209
- DOI: 10.1073/pnas.061500998
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Comment
VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation
Proc Natl Acad Sci U S A.
.
Abstract
The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.
Figures
The high chlorophyll fluorescence mutant hcf155 is
deficient in function and structural integrity of thylakoid membranes.
(a) Measurements of chlorophyll fluorescence induction
and P700 redox kinetics of hcf155 mutant and wild-type
plants of Arabidopsis thaliana. The minimal level of
fluorescence (Fo) of dark-adapted whole plants was measured by
switching on a pulsed measuring beam of red light. A saturating pulse
of white light was then applied to determine the maximum level of
fluorescence in the dark-adapted state (Fm). Subsequently actinic
(white) light was switched on to drive photosynthesis. A further
saturating flash allowed us to measure the fluorescence maximum in the
light (Fm′). The activity of photosystem I was investigated by
measuring absorbance changes of P700 at 830 nm
(Amax) induced by far-red light (720 nm).
Plants were preilluminated for 4 min with actinic light. Far-red light
was switched on to oxidize P700. A saturating pulse of white light was
then applied to rereduce P700 and thus to determine
Amax. (b) Immunoblot analysis
of representative photosynthetic proteins in hcf155
mutant and wild-type (WT) plants. Total membrane proteins were isolated
from mutant and wild-type plants, separated on SDS/polyacrylamide
gels, and transferred to nitrocellulose membranes. Immunoblots were
performed with antisera made against RbcL, the large subunit of
ribulose-1,5-bisphosphate carboxylase/oxygenase; PsaD, subunit D of
photosystem I; PsbA/D, subunits D1 and D2 of photosystem II; and
PetA, cytochrome f. Fifteen micrograms of total protein
(WT and hcf155) or, in the case of the wild-type
extract, corresponding dilutions were analyzed. (c)
Electron micrographs showing the ultrastructure of chloroplasts from
mutant leaves of progressing age. (Top) An electron
micrograph of a typical wild-type chloroplast. (Scale bars represent 1
μm.)
The T-DNA insertion in hcf155 reduces the
expression of a gene [gene designation VIPP1
(AAC27135.1)] that encodes a chloroplast protein that is located in
both the inner envelope and the thylakoid membrane. (a)
Localization of the T-DNA insertion site and identification of the
affected gene by Northern blot analysis. The hybridization probes
specific for the AAC27134 and AAC27135 genes that flank the T-DNA
insertion were generated by PCR. RNA loadings were 10 μg of total RNA
(WT and hcf155) and a 50% dilution in the case of
wild-type RNA. (b) Amino acid alignment of the
VIPP1/IM30 precursor proteins of Arabidopsis thaliana
(At) and Pisum sativum (Ps). The putative cleavage sites
are indicated. (c) Localization of the VIPP1 protein in
plastid fractions of A. thaliana wild-type plants with
the use of marker proteins of established topology: Tic40, subunit of
the protein translocation machinery of the inner envelope (IE) (29);
Toc160, subunit of the protein translocation machinery of the outer
envelope (OE) (16); RbcL, large subunit of the stromal (STR) enzyme
ribulose-1,5-bisphosphate carboxylase/oxygenase; LHCP, major
chlorophyll a/b-binding antenna protein
of photosystem II, a thylakoidal (THY) protein complex. Equal amounts
of protein (8 μg per lane) were loaded onto the gel.
A full-size cDNA encoding the VIPP1 protein of A.
thaliana and driven by the cauliflower 35S promoter complements
the hcf155 mutant phenotype. (a)
Chlorophyll fluorescence traces of wild-type and hcf155
homozygous mutant plants are shown for comparison. (b)
Electron micrographs of chloroplasts from complemented mutant plants in
comparison with wild-type and homozygous hcf155 mutant
plants. (Scale bars represent 1 μm.)
hcf155 mutant plants are deficient in the budding of
vesicles from the inner envelope membrane. Representative electron
micrographs are shown. The formation of vesicles can be observed in
leaves from wild-type plants incubated at 4°C for 45 min, but not at
22°C. Vesicle budding is lacking in homozygous hcf155
mutant seedlings but not in the photosystem II mutant
hcf136 (20). Complemented hcf155 mutant
regains the ability to form vesicles. (Scale bars represent 1 μm.)
Comment on
-
Discovery of a protein required for photosynthetic membrane assembly.Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):3633-5. doi: 10.1073/pnas.071056598. Proc Natl Acad Sci U S A. 2001. PMID: 11274378 Free PMC article. No abstract available.
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