Mosquito clathrin heavy chain: analysis of protein structure and developmental expression in the ovary during vitellogenesis.
We have deduced the amino acid sequences of clathrin heavy chain (CHC) polypeptides based on cDNA and genomic clones from the mosquito, Aedes aegypti. Two isoforms which differ in the very beginning of the N-terminal domain, ovary-specific AaCHCa and somatic-specific AaCHCb, were identified, characterized and compared to one another as well as to CHC polypeptides from different species. The 1682 amino acid sequence of the AaCHCa isoform predicts a molecular mass (M[r]) of 191,743 daltons and an isoelectric point of 5.80, whereas the 1674 amino acid sequence of the AaCHCb isoform predicts a M(r) of 191,033 daltons and an isoelectric point of 5.71. Both mosquito AaCHC isoforms are highly conserved, with full-sequence identities of 88% to Drosophila melanogaster, 81% to mammal (rat, cow and human), 71% to C. elegans, 58% to Dictyostelium 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 decreasing conservation through the N-terminal domain, trimerization domain, and the relatively diverged C-terminal region. The protein domains do not directly correspond to specific exons of the mosquito AaCHC gene, with the exception of exon 6 which encodes the C-terminal domain of 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 AaCHC polypeptide localized it to the coat of coated pits and coated vesicles in oocytes from vitellogenic follicles. Northern blot and in situ hybridization analyses suggest that regulation of AaCHC gene expression in the ovary is complex, and it likely involves both developmental and hormonal signals.
Kokoza
V A
VA
Department of Entomology, Michigan State University, East Lansing 48824-1115, USA.
Snigirevskaya
E S
ES
Raikhel
A S
AS
D000330:Aedes / Q000235:genetics* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002966:Clathrin / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D033922:Clathrin Heavy Chains; D003034:Coated Pits, Cell-Membrane / Q000737:chemistry; D018699:Coated Vesicles / Q000737:chemistry; D017124:Conserved Sequence / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D017344:Genes, Insect / Q000235:genetics; D008969:Molecular Sequence Data; D009865:Oocytes / Q000737:chemistry; D010053:Ovary / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D014818:Vitellogenesis / Q000235:genetics*
D002966:Clathrin; D012333:RNA, Messenger; D033922:Clathrin Heavy Chains