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

113 Functional analysis of AtSDK1 and AtSKD1-interacting proteins in the endosomal pathways
of Arabidopsis
Thomas Haas, Marek Sliwinski, Marisa Otegui
Dept. of Botany, University of Wisconsin, 430 Lincoln Drive, Madison, WI 53706, USA
Endosomal compartments play a fundamental role in the sorting, recycling, and turnover of membrane proteins, the
downregulation of receptors, and the trafficking of proteins to vacuoles and lysosomes in all eukaryotic cells. In addition,
endosomal trafficking plays a essential role in various aspects of plant life, such as development, cell type specification,
cell wall remodeling, gravitropic response, signal transduction, and hormone transport. Endosomal trafficking is regulated
by a complex molecular machinery. Yeast Vps4p and mammalian SKD1 are AAA ATPases required for the endosomal
sorting of secretory and endocytic cargo. We have identified a putative SKD1 homolog in Arabidopsis called AtSKD1.
By immunogold labeling and expression of fluorescent fusion proteins, we have determined that SKD1 localizes to
the cytoplasm and to multivesicular endosomes. In addition, GFP-SKD1 partially colocalizes with fuorescent fusion
proteins of well characterized endosomal Rab GTPases such as AtRabF2b and RabF2a and with the endocytic markers
FM4-64 and FM5-95. The expression of AtSKD1 E232Q , an ATPase-deficient version of AtSKD1, induces alterations in
the vacuolar and endosomal systems of tobacco BY2 cells and ultimately leads to cell death. We have performed a yeast
two-hybrid screening to identify AtSKD1-interacting proteins in Arabidopsis. One of the identified interactors, AtLIP5,
is a homologue of mammalian LIP5/SBP1 and yeast Vta1p, which are known to be positive regulators of SKD1/Vps4p
ATPase activity (Azmi et al. 2006 J. Cell Biol. 172, 705-717). We have isolated a knock out homozygous mutant line
with a T-DNA insertion in AtLIP5. Although these mutant plants are viable, they exhibit reduced growth and produce
fewer seeds. We are analyzing the subcellular localization patterns of AtSKD1 and AtLIP5 in the context of their putative
interactions.
114 MscS-Like Proteins in Arabidopsis: Plastid Morphology and More
Elizabeth Haswell, Elliot Meyerowitz
Caltech
A fundamental question in biology is how cells perceive mechanical stimuli. Recent progress in the field of
mechanotransduction has revealed that animal and bacterial cells sense sound, touch, and osmotic pressure through the
action of mechanosensitive (MS) ion channels. Plants also respond to mechanical stimuli, and numerous mechanosensitive
ion channel activities have been identified in plant membranes; however, the proteins responsible for these phenomena
have not been identified. We have initiated the mechanistic and genetic characterization of a family of Arabidopsis
proteins related to the bacterial mechanosensitive ion channel MscS, hypothesizing that they may be involved in
mechanotransduction. Two Arabidopsis MscS-Like (MSL) proteins, MSL1 and MSL3, are capable of protecting bacteria
from lysis upon hypoosmotic downshock, suggesting that at least some members of the family are mechanically gated
ion channels. Results from subcellular localization and reporter gene experiments illustrate that the MSL protein family
has evolved to function in the chloroplast and mitochondrial envelopes, as well as in other cellular membranes, and
that family members are expressed in cells that undergo large, dynamic changes in turgor pressure. We have isolated
insertional mutants in MSL2 and MSL3 and shown that msl2-1; msl3-1 double mutants have misshapen and enlarged
plastids. Furthermore, MSL2- and MSL3-GFP fusion proteins are localized to the plastid envelope in discrete foci, and
co-localized with the plastid division protein AtMinE (Haswell and Meyerowitz, Current Biology 16:1-11, 2006). The
insertion in the msl3-1 mutant is predicted to produce a truncated protein that lacks the last 162 amino acids; we now
find that GFP fused to a similarly truncated version of MSL3 is not localized to discrete foci but is evenly distributed
around the plastid envelope. This finding suggests that localization to foci requires a specific domain in the C-terminus
of MSL3, and that disruption of this domain may play a role in the plastid phenotype of the msl2-1; msl3-1 double
mutant. Results from our ongoing characterization of the genetic and interactions between MSL2, MSL3 and previously
identified plastid division genes will also be presented.