We have previously isolated epsilon-COP, the alpha-COP interactor in COPI of Aspergillus nidulans, by yeast two-hybrid screening. To understand the function of epsilon-COP, the aneA+ gene for epsilon-COP/AneA was deleted by homologous recombination using a gene-specific disruption cassette. Deletion of the epsilon-COP gene showed no detectable changes in vegetative growth or asexual development, but resulted in decrease in the production of the fruiting body, cleistothecium, under conditions favorable for sexual development. Unlike in the budding yeast Saccharomyces cerevisiae, in A. nidulans, over-expression of epsilon-COP did not rescue the thermo-sensitive growth defect of the alpha-COP mutant at 42degrees C. Together, these data show that epsilon-COP is not essential for viability, but it plays a role in fruiting body formation in A. nidulans.
Aspergillus nidulans* ; Coatomer Protein* ; Fruit ; Homologous Recombination ; Mass Screening ; Saccharomyces cerevisiae ; Saccharomycetales ; Sexual Development ; Yeasts

Studies on coat protein I (COPI) have contributed to a basic understanding of how coat proteins generate vesicles to initiate intracellular transport. The core component of the COPI complex is coatomer, which is a multimeric complex that needs to be recruited from the cytosol to membrane in order to function in membrane bending and cargo sorting. Previous structural studies on the clathrin adaptors have found that membrane recruitment induces a large conformational change in promoting their role in cargo sorting. Here, pursuing negative-stain electron microscopy coupled with single-particle analyses, and also performing CXMS (chemical cross-linking coupled with mass spectrometry) for validation, we have reconstructed the structure of coatomer in its soluble form. When compared to the previously elucidated structure of coatomer in its membrane-bound form we do not observe a large conformational change. Thus, the result uncovers a key difference between how COPI versus clathrin coats are regulated by membrane recruitment.
ADP-Ribosylation Factor 1 ; chemistry ; metabolism ; Animals ; Coatomer Protein ; chemistry ; metabolism ; Cytosol ; chemistry ; metabolism ; GTPase-Activating Proteins ; chemistry ; metabolism ; Humans ; Membranes, Artificial ; Rats

We have cloned a alpha-COP homolog, ancop, from Aspergillus nidulans by colony hybridization of chromosome specific library using alpha-COP homologous fragment as a probe. The probe DNA was amplified with degenerated primers designed by comparison of conserved region of the amino acid sequences of Saccharomyces cerevisiae alpha-COP, Homo sapiens HEP-COP, and Drosophila melanogaster alpha-COP. Full length cDNA clone was also amplified by RT-PCR. Comparison of genomic DNA sequence with cDNA sequence obtained by RT-PCR revealed 7 introns. Amino acid sequence similarity search of the anCop with other alpha-COPs gave an overall identity of 52% with S. cerevisiae, 47% with human and bovine, 45% with Drosophila and Arabidopsis . In upstream region from the transcription start site, a putative TATA and CAAT motif were also identified.
Amino Acid Sequence ; Arabidopsis ; Aspergillus nidulans* ; Aspergillus* ; Base Sequence ; Clone Cells ; Coatomer Protein* ; DNA ; DNA, Complementary ; Drosophila ; Drosophila melanogaster ; Humans ; Introns ; Saccharomyces cerevisiae ; Sequence Homology ; Transcription Initiation Site