15th International Conference on Arabidopsis Research - TAIR

T03-023
Systematic determination of protein localisation in
Arabidopsis cells
Matthew Tomlinson(1), Olga Koroleva(1), Peter Shaw(1), John Doonan(1)
1-John Innes Centre, Norwich, UK
We have developed and applied a novel streamlined approach for studying
intracellular localisation of GFP-fusion proteins in Arabidopsis cell suspensions.
High throughput transient transformation of cell suspensions by
constructs containing constitutive promoters and a N-terminal GFP fusion,
delivered by a hypervirulent strain of Agrobacterium, allowed processing of
large batches of constructs. A set of Arabidopsis full-length trimmed ORF
clones (obtained from the SSP consortium, http://signal.salk.edu/SSP/index.
html) have been used for semi automated cloning to convert to GATEWAY expression
vectors in a 96 well format. The selection of ORFs has been biased
towards genes involved in regulation of growth and development.
Localisation patterns of 155 expressed fusion proteins have been studied
and the patterns classified into five main categories: nucleus, nucleolus,
cytoplasm, organelles and endomembrane compartments, and cell wall. Representative
members of several functional groups of proteins included protein
kinases (CDK and cyclins, MAPK), protein phosphatases, transcription factors
and DNA-associated proteins (AP2, GTFII, HAP, WRKY, MYB, bHLH, DRT families),
RNA processing proteins, translation factors, metabolic enzymes and
signal transduction factors. Several genes annotated in GenBank as unknown
have been ascribed a protein localisation pattern. The localisation patterns
for a number of functional groups of proteins were compared with the bioinformatic
predictions of subcellular target domains based on the amino acid
sequence motifs (using PSORT software). The set of proteins analysed so
far have provided some interesting insights into possible biological functions,
and in some cases have indicated regulation by control of localisation. This
approach can be extended for functional studies including precise cellular
localisation and prediction of function of unknown proteins, confirmation of
bioinformatic predictions, co-localisation and FRET analysis of interactions,
and proteomic experiments.
T03 Cell Biology
T03-024
Chloroplast division site placement requires
dimerisation of the ARC11/AtMinD1 protein in
Arabidopsis
Makoto Fujiwara(1, 2), Ayako Nakamura(2), Ryuuichi Itoh(2), Yukihisa Shimada(2),
Shigeo Yoshida(2), Simon Geir Møller(1)
1-Department of Biology, University of Leicester, University Road, Leicester LE1 7RH, United
Kingdom
2-Plant Functions Laboratory and Plant Science Center, RIKEN, Hirosawa 2-1, Wako, Saitama
351-0198, Japan
Chloroplast division is mediated by the coordinated action of a prokaryotederived
division system(s) and a host eukaryote-derived membrane fission
system(s). The evolutionary conserved prokaryote-derived system comprises
several nucleus-encoded proteins two of which are thought to control
division site placement at midpoint of the organelle: a stromal ATPase MinD
and a topological specificity factor MinE. Here, we show that arc11, one of
12 recessive accumulation and replication of chloroplasts (arc) mutants in
Arabidopsis, contains highly elongated and multiple-arrayed chloroplasts in
developing green tissues. Genomic sequence analysis revealed that arc11
contains a missense mutation in alpha-helix 11 of the chloroplast-targeted
AtMinD1 changing an Ala at position 296 to Gly (A296G). Introduction of
wild-type AtMinD1 restores the chloroplast division defects of arc11 and
quantitative RT-PCR analysis demonstrated that the degree of complementation
was highly dependent on transgene expression levels. Overexpression
of the mutant ARC11/AtMinD1 in transgenic plants results in inhibition of
chloroplast division demonstrating that the mutant protein has retained its
division inhibition activity. However, in contrast to the defined and punctate
intraplastidic localization patterns of an AtMinD1-YFP fusion protein, the
single A296G point mutation in ARC11/AtMinD1 results in aberrant localization
patterns inside chloroplasts. We further show that AtMinD1 is capable
of forming homodimers and that this dimerisation capacity is abolished by
the A296G mutation in ARC11/AtMinD1. Our data demonstrate that arc11
is a loss-of-function mutant of AtMinD1 and suggest that the formation
of functional AtMinD1 homodimers is paramount for appropriate AtMinD1
localization ultimately ensuring correct division machinery placement and
chloroplast division in plants.
Fujiwara, M.T., Nakamura, A., Itoh, R., Shimada, Y., Yoshida, S. and Møller, S.G. (2004). J Cell Sci.
117. 2399-2410.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin