75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

131 Sub-Cellular Localization of DRG Family Protein
Benjamin Nelson, Joel Stafstrom
Department of Biological Sciences, Northern Illinois University, Dekalb, IL 60115
DRGs (Developmentally Regulated G-Proteins) were first discovered in embryonic mouse brains. All eukaryotes
appear to have two Drg genes (Drg1 and Drg2), Archaea have a single gene, and bacteria have one Obg gene, which
encodes a related G-protein. DRG proteins are very highly conserved. Amino acid identity among fungal, animal and
plant DRG1s is about 71%, and AA identity of DRG2s is nearly as high. Arabidopsis DRG1 (At4g39520) and DRG2
(At1g17470) are about 57% identical. Arabidopsis DRG3 (At1g72660) is 95% identical to DRG2, suggesting a recent gene
duplication. This level of conservation suggests that these proteins perform an important cellular activity, yet relatively
little is known about the function of these proteins. Single-gene knock-outs of these genes, produced both by T-DNA
insertion and RNA interference, appear quite normal when grown under normal conditions. Double mutants are being
generated. Growth and development under a variety of stress conditions is being analyzed as well. A variety of techniques
are being used to determine patterns of subcellular localization, including standard biochemical fractionation, affinity
pull-down assays, and GFP fusions in transgenic plants. Differential and rate-zonal centrifugation demonstrates that
DRG1 and DRG2 co-localize with various ribosome populations. However, the specific localization patterns are unique,
suggesting a distinct function for each protein. We have also found that the full-length 45kd DRG2 protein is processed
to 43kd and 30kd forms, each of which is found in specific subcellular fractions. Affinity pull-down assays using FLAGtagged
ribosomes, in both Arabidopsis and Saccharomyces, also indicate that the DRG proteins are ribosome-associated.
GFP studies, while in the early stages, indicate a cytosolic and possibly nuclear localization for the DRG proteins.
132 A Mutation in Translocon of Outer Membrane of Chloroplasts 132 (Toc132) Enhances the
Gravitropic Defect of the altered response to gravity 1 (arg1) Mutant
John Stanga 1 , Kanok Boonsirichai 2 , John Sedbrook 3 , Patrick Masson 1
1
University of Wisconsin-Madison, 2 Center for Atomic Research for Peace Purposes, Bangkok, Thailand ,
3
Illinois State University, Normal, Illinois
Arabidopsis roots perceive gravity and reorient their growth accordingly. The root cap is necessary for a full response
to gravity. Starch-dense amyloplasts within the columella cells of the root cap are important for gravitropism, as starchless
mutants (pgm1) display an attenuated response to gravistimulation. However, our understanding of the molecular events
controlling this behavior is incomplete. The altered response to gravity 1 (arg1) mutant is involved with the early phases of
gravity signal transduction and is in a genetically distinct pathway from pgm1. arg1 roots and hypocotyls respond slowly
to gravistimulation. Expression of ARG1 in the root cap is sufficient to rescue the root gravitropism defect specifically,
while expression of ARG1 in the endodermis is sufficient to rescue the hypocotyl defect specifically. arg1 seeds were
mutagenized with EMS to identify new mutants that would enhance the gravitropic defect of arg1 and therefore be
potential members of the PGM1 pathway. The roots of one such mutant, mar2, grow in random directions only when
arg1 is present, yet have no other obvious defects. Also, mar2 amyloplasts appear normal when observed by electron
microscopy. mar2 plants possess a mutation in the Translocon of Outer Membrane of Chloroplast 132 (TOC132) gene,
At2g16640, that results in premature termination of translation. Overexpression of TOC132 rescues the random growth
phenotype of arg1mar2 roots. In addition to green tissues, Toc132 is expressed in tissues that lack chloroplasts, such
as the columella cells of the root cap. Toc132 is thought to act as a receptor component of the Toc complex. Its exact
contribution to the determination of root growth direction remains the subject of ongoing endeavors.