Page 169 - Plant Canada 2024 Proceeding

PLANT CANADA 2024

secretion process. Different from the other classes of molecular chaperones within ER, the HSP90 family
heat short proteins are present, but only in higher multicellular organisms suggesting a critical role of ER-
localized HSP90 family protein in cell differentiation and cell-to-cell communications. In our lab, we are
interested in the structure and function of the specific ER-localized HSP90.7 from Arabidopsis thaliana.
We identified that the protein contains unique structural elements that regulate the protein’s chaperone
activity and play critical roles in ER-specific stress resistance. We also aimed to understand whether the
plant ER-localized HSP90.7 has a different mechanism of action from its animal orthologs. Additionally,
we screened and identified an HSP90.7 knockout mutant line that showed a seedling lethality phenotype
together with defective trichomes development, improper chloroplast functionality, and impaired apical
meristem maintenance and differentiation. Comparative transcriptome and proteome analyses revealed
roles of the protein in a multitude of cellular processes. Particularly, we measured a much-reduced auxin
content in both root and shoot tissues and then investigated how the cellular auxin biosynthesis and
transport systems are impacted when the HSP90.7 protein is missing. This study therefore not only
fulfilled a gap in understanding the essential role of HSP90 paralogs in eukaryotes, but also provided a
mechanistic insight on the ER localized chaperone in regulating plant growth and development via
modulating cellular auxin homeostasis.

[O131] ARABIDOPSIS ICK/KRP CYCLIN-DEPENDENT KINASE INHIBITORS ARE INTRINSICALLY
DISORDERED PROTEINS AND REGULATED BY BOTH UBIQUITIN-DEPENDENT AND UBIQUITIN-
1
1
1,2
1
INDEPENDENT MECHANISMS. Shengjian Ye , Sheng Wang , Ron Chan , Ling Cao , and Hong
1 1
Wang . Dept. of Biochemistry, Microbiology & Immunology, University of Saskatchewan, Saskatoon SK,
S7N 5E5, Canada; and Present address: Aquatic and Crop Resource Development, National Research
2
Council of Canada, Saskatoon, SK, S7N 0W9, Canada
Correspondence to: hong.wang@usask.ca

Plants have a family of cyclin-dependent kinase (CDK) inhibitors called ICKs (interactors/inhibitors of
CDK) (or KRPs, Kip-related proteins). ICK proteins have important functions in cell proliferation,
endoreduplication, plant growth and reproductive development. Since the functions of ICKs depend on
the protein levels, it is crucial to understand how ICK protein levels are regulated. It has been shown
previously that the ubiquitin (Ub) proteasome system (UPS) has a major role in the degradation of ICKs.
However, little is known regarding the specific signal sequences that confer instability to ICKs. Using
various computational tools including AlphaFold2, we have shown that ICK proteins are mostly disordered
and unstructured except for the conserved C-terminal region. Experimentally, we observed consistently
that all ICK proteins ran slower than their expected molecular sizes in gel electrophoresis, which is a
feature observed on intrinsically disordered proteins (IDPs). These results suggest that ICKs are IDPs. To
identify sequence signals responsible for ICK instability and degradation, we fused various Arabidopsis
ICK sequences to the green fluorescent protein (GFP), and determined their effects on the fusion proteins
in plants, yeast and E. coli. The N-terminal regions of ICKs drastically reduced GFP fusion protein levels
in Arabidopsis plants. A number of short sequences of 10 – 20 residues were found to decrease GFP
fusion protein levels, when fused at the N-terminus or C-terminus. Three of the four short sequences from
ICK3 showed a similar function in yeast. Intriguingly, three short sequences from ICK1 and ICK3 caused
the degradation of the fusion proteins in E. coli. We thus have identified a number of short protein-
destabilizing sequences. In addition to the established Ub-dependent protein degradation, the finding that
some of the short sequences still show the protein-destabilizing property in E. coli suggests that they
function through a Ub-independent mechanism since E. coli does not have the major components of the
eukaryotic UPS system. The present results provide new insight regarding how ICKs are regulated.
Importantly, since an estimated 25-30% of the eukaryotic proteomes contain long intrinsically disordered
regions, the present results may provide useful leads for studying many other unstable proteins
containing intrinsically disordered regions.

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