Va. Kokoza et al., MOSQUITO CLATHRIN HEAVY-CHAIN - ANALYSIS OF PROTEIN-STRUCTURE AND DEVELOPMENTAL EXPRESSION IN THE OVARY DURING VITELLOGENESIS, Insect molecular biology, 6(4), 1997, pp. 357-368
We have deduced the amino acid sequences of clathrin heavy chain (CHC)
polypeptides based on cDNA and genomic clones from the mosquito, Aede
s aegypti. Two isoforms which differ in the very beginning of the N-te
rminal domain, ovary-specific AaCHCa and somatic-specific AaCHCb, were
identified, characterized and compared to one another as well as to C
HC polypeptides from different species. The 1682 amino acid sequence o
f the AaCHCa isoform predicts a molecular mass (M-r) of 191,743 dalton
s and an isoelectric point of 5.80, whereas the 1674 amino acid sequen
ce of the AaCHCb isoform predicts a M-r of 191,033 daltons and an isoe
lectric point of 5.71. Both mosquito AaCHC isoforms are highly conserv
ed, with full-sequence identities of 88% to Drosophila melanogaster, 8
1% to mammal(rat, cow and human), 71% to C. elegans, 58% to Dictyostel
ium discoideum, and 49% to yeast CHC polypeptides. The highest degree
of conservation is in the middle portion of the mosquito CHC molecule
which includes the linker region and extended triskelion arm, with dec
reasing conservation through the N-terminal domain, trimerization doma
in, and the relatively diverged C-terminal region. The protein domains
do not directly correspond to specific exons of the mosquito AaCHC ge
ne, with the exception of exon 6 which encodes the C-terminal domain o
f the CHC polypeptide. Polyclonal antibodies raised against a bacteria
-expressed AaCHC fusion protein recognized one major band of about 180
kDa in vitellogenic ovary whole-lysate. Immunogold labelling of the A
aCHC polypeptide localized it to the coat of coated pits and coated ve
sicles in oocytes from vitellogenic follicles. Northern blot and in si
tu hybridization analyses suggest that regulation of AaCHC gene expres
sion in the ovary is complex, and it likely involves both developmenta
l and hormonal signals.