Program of the 2001 International Worm Meeting - Sternberg Lab ...

551
551. A Caenorhabditis elegans
catalase with an unusual feature
Summanuna H. Togo 1 , Motohiro
Maebuchi 1 , Masanori Bun-ya 1 ,
Sadaki Yokota 2 , Tatsuyuki Kamiryo 1
1 Hiroshima University, Kagamiyama 1-7-1,
Higashi-Hiroshima, Japan, 739-8521
2 Yamanashi Medical University, Biological
Laboratory, Nakakuma, Tamaho, Japan
409-3898
Earlier attempts to detect peroxisomes in the
free-living nematode Caenorhabditis elegans by
the alkaline-diaminobenzidine method that
exploits the peroxidase activity of only
peroxisomal catalase at pH 9 were not
successful. This motivated us to purify catalase,
a peroxisomal marker form C. elegans whose
genome has genes ctl-1 and ctl-2 encoding
catalases closely similar to each other. A near to
homogeneity preparation was obtained with a
purification fold of 2900 and a yield of 15%.
The purified enzyme is a protein of molecular
weight of 220kDa and is composed of four
subunits. Antiserum raised against a synthetic
pentadecapeptide corresponding to the
C-terminal sequence of the ctl-2 product
specifically recognized the purified enzyme, and
was used in immunoelectron microscopy to
confirm the peroxisomal localization of catalase
obtained in cell fractionation. The enzyme is
similar in squence to other catalases from
various sources, except that it showed an
unexpectedly low pH optimum (pH 4.0) for its
peroxidase activity. This feature is different
from catalases thus far known and explains why
the C. elegans peroxisome was undetectable
when the widely used
alkaline-diaminobenzidine staining method was
applied.
552. Biochemical and functional
analysis of SKP1 family in
Caenorhabditis elegans
Atsushi Yamanaka 1,2 , Masayoshi
Yada 1 , Hiroyuki Imaki 1 , Yasumi
Ohshima 3 , Kei-ichi Nakayama 1,2
552
1 Department of Molecular and Cellular
Biology,Medical Institute of
Bioregulation,Kyushu
University,Maidashi,Fukuoka,812-8582,JAPAN
2 CREST,JST
3 Department of Bioligy,Faculty of
Sciences,Kyushu University Graduate
School,Hakozaki,Fukuoka 812-8581,Japan
The ubiuquitin-proteasome pathway is a key
mechanism for substrate-specific degradation to
control the abundance of a number of proteins.
SCF complex, one of ubiquitin-protein ligases
(E3s), regulates cell cycle progression, signal
transduction, and many other biological
systems. The SCF complex consists of
invariable components, such as Skp1, Cul-1 and
Rbx1, and variable components called F-box
proteins that bind to Skp1 through the F-box
motif. F-box proteins are substrate-specific
adaptor subunits that recruit substrates to the
SCF complex. Surprisingly, we found that the
genome of Caenorhabditis elegans (C. elegans)
contains at least 20 Skp1-like sequenses,
whereas one or a few Skp1 is present in humans.
Therefore, we studied C. elegans Skp1-like
proteins (CeSkp1) that are likely to be variable
components of SCF complex in addition to
F-box proteins. At least, seven CeSkp1s were
associated with C. elegans Cul-1 (CeCul-1) in
yeast two-hybrid system as well as
co-immunoprecipitation assay in mammalian
cells, and these expression patterns were
different in C. elegans. By RNA interference
(RNAi), two of these CeSkp1s showed
embyonic lethality and four showed the
phenotype of slow growth. There were
differences among CeSkp1s in ability to interact
with F-box proteins. These results suggest that
CeSkp1s, like F-box proteins, act as variable
components of SCF complex in C. elegans.