SUMO (small ubiquitin-like modifier) protein, also known as Smt3
(suppressor of Mif2 protein 3) of Saccharomyces cerevisiae is an
ubiquitin-like protein due to the similar post-transcriptional modifications
to their substrates. There are three members of SUMO genes (SUMO-1,
SUMO-2 & SUMO-3) in the vertebrate, while only one SUMO gene exists
in the invertebrate. Covalent modification of cellular proteins by the
SUMO regulates various cellular processions, such as nuclear transport,
transcription repression and cellular apoptosis. To investigate the biological
functions of SUMO-1 and SUMO-2, yeast two hybrid assays were applied.
Results showed that N-terminus (Daxx1, 1-282 amino acids) and
C-terminus (Daxx4, 607-740 amino acids) of Daxx were the SUMOs
interacting fragments.
For identification of the sumoylation site on Daxx1 and Daxx4, six
mutants (K60R, K630A, K631A, K634A, K630, 631A and K630, 631,
634A) were constructed. In vitro sumoylation were applied in the Daxx1
fragment and mutated Daxx1 (K60R) as well as the Daxx4 fragment and
mutated Daxx4 (K630A, K631A , K634A, K630, 631A and K630, 631,
634A) to identify the sumoylation sites of Daxx.
Our results showed that Daxx1 K60 was one of the sumoylation sites,
neverthless it was not a major sumoylation site. The major sumoylation
sites were on the C-terminus of Daxx (Daxx4). The major sumoylation sites
of SUMO-1 on Daxx4 seemed different from those of SUMO-2. Mutants
(K631A and K634A) of Daxx4 decreased the yields of sumoylation
complexes of SUMO-1 more than that of Daxx4 K630. However, mutants
(K630A and K631A) of Daxx4 decreased the yields of sumoylation
complexes of SUMO-2 more than that of Daxx4 K634. Thus we propose
that the major sumoylation sites of SUMO-1 on Daxx are K631A and
K634A and that of SUMO-2 are K630A and K631A. Daxx may have other
sumoylation sites on the Daxx C-terminal 635-740 amino acids fragment,
unless the sumoylation reactions of Daxx mutants were pseudo-positive
reactions which might be caused by the improper folding of Daxx4 during
in vitro sumoylation.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0203104-140623 |
| Date | 03 February 2004 |
| Creators | Huang, Yi-hsin |
| Contributors | Yi-Ren Hong, Angela Chen, Ming-Hong Tai, S. S.-L. Li |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0203104-140623 |
| Rights | not_available, Copyright information available at source archive |
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