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

123 The Arabidopsis CDC48 Adapter Protein PUX2 Plays a Role in ER Membrane Organization
David Rancour, Sookhee Park, Lisa Koch, Sebastian Bednarek
Biochemistry Department, University of Wisconsin-Madison, Madison, WI 53706
CDC48/p97 is a conserved and essential hexameric AAA-ATPase that functions as a molecular chaperone in
numerous diverse cellular activities. CDC48/p97 activity is recruited to specific functions through its interaction with
adapter proteins. Our working hypothesis is that AtCDC48 and the Arabidopsis homolog of syntaxin5/Sed5p, SYP31,
mediates events important for plant cytokinesis. Using affinity chromatography and MALDI-TOF mass spectrometry
we have identified two uncharacterized plant UBX-domain containing proteins, PUX1 and PUX2, which interact with
AtCDC48. UBX-domains are ubiquitin-like protein folds that function as interaction domains for CDC48/p97. The
Arabidopsis genome encodes 15 PUX proteins. All PUX proteins tested so far interact with AtCDC48 suggesting this
to be an AtCDC48 regulatory protein family.
Characterization of PUX2 indicates that it is a peripheral membrane protein that interacts with AtCDC48 in vitro
and co-fractionates with membrane-associated but not soluble AtCDC48 in vivo. Biochemical reconstitution and
immunolocalization data suggest that PUX2 facilitates the interaction of SYP31 and AtCDC48 during interphase and
cytokinesis, thereby regulating an AtCDC48 membrane-associated function. Deletion analysis of PUX2 protein domains
reveals the requirement of its PUG domain and not its UBX-domain for interaction with AtCDC48. Sequence analysis of
the UBX-domain from PUX2 suggests that divergence in amino acid composition might be responsible for the inability
of the domain to interact with AtCDC48. Furthermore, analysis of protein domains of AtCDC48 required for interaction
with PUX2 show that PUX2 binds AtCDC48 through the C-terminal D2 domain, an unconventional interaction for
CDC48/p97 adapters. These studies provide the first evidence that the PUG domain may be an alternate interaction domain
for AtCDC48. Loss-of-function pux2-1 Arabidopsis plants display altered endoplasmic reticulum (ER) distribution and
a corresponding increase in cellular chloroplast content. These data taken together suggest a role for PUX2, AtCDC48
and SYP31 in maintaining ER membrane function. Models of PUX2 function will be presented.
124 Characterization of an Ectopic Cell Separation Mutant tfa1 'Things Fall Apart'
HONGYU RAO, Cory Hirsch, Joonyup Kim, Sara Patterson
Horticulture Department, University of Wisconsin at Madison
Although we often think of the plant cell wall as rigid and inflexible, it is dynamic, responding to developmental and
environmental cues, and this plasticity is essential to basic plant development. Processes such as cell to cell adhesion
and cell separation are examples of this dynamic nature of the plant cell wall; however, little has been studied about how
plants integrate the processes of cell to cell adhesion and plant cell separation with developmental and environmental
cues. We have identified several ectopic cell separation mutants that we have designated tfa "things fall apart." These
mutants are all characterized by unregulated cell separation in epidermal, cortical and vascular tissues. This process is
most severe in young seedlings affecting the hypocotyl, cotyledon, and first true leaves. Most of the young seedlings
undergoing the irregular ectopic cell separation usually can not develop normal root and shoot system and eventually
die when cultured on 1/2MS media with 0.8% agar. Crosses among the mutants indicate that two of these genes are
allelic and recessive mutations, and we have designated them tfa1-1 and tfa1-2. Additional characterization of tfa1-2 has
demonstrated that the defects in cell adhesion in tfa1-2 young seedlings can be partially avoided by culturing tfa1-2 seeds
on the 1/2 MS media with a higher percentage agar or at low temperatures. Expression of several cell wall associated
genes has been analyzed by RT-PCR and several of these genes show changes in expression in the mutant background.
Microarray analysis of tfa1-2 has been conducted and there are numerous cell wall associated genes as well as others
that are strongly upregulated or down regulated. tfa 1-2 was identified from an EMS screen of Arabidopsis seedlings
and PCR-based mapping indicates that it is positioned on the right arm of Chromosome I. We will present phenotypic,
physiological and molecular characterization of tfa1. We propose that there are several regulatory pathways affecting
cell separation programs in the plant. Our studies on TFA1 will contribute to our understanding of how plants regulate
cell to cell adhesion and cell separation processes during plant development.
This work was funded by USDA grant 0035301-9085 and NSF DBI
*Corresponding author: spatters@wisc.edu or hrao@wisc.edu