In this research we report that the sepG1 mutation in Aspergillus nidulans resides in gene AN9463, which is predicted to encode an IQGAP orthologue. The genetic lesion is predicted to result in a G-to-R substitution at residue 1637 of the 1737-residue protein in a highly conserved region of the RasGAP-C-terminal (RGCT) domain. When grown at restrictive temperature, strains expressing the sepG^G1637R (sepG1) allele are aseptate, with reduced colony growth and aberrantly formed conidiophores. The aseptate condition can be replicated by deletion of AN9463 or by downregulating its expression via introduced promoters. The mutation does not prevent assembly of a cortical contractile actomyosin ring (CAR) at putative septation sites, but tight compaction of the rings is impaired and the rings fail to constrict. Both GFP::SepG wild type and the GFP-tagged product of the sepG1 allele localize to the CAR at both permissive and restrictive temperatures. Downregulation of myoB (encoding the A. nidulans type-II myosin heavy chain) does not prevent formation of SepG rings at septation sites, but filamentous actin is required for CAR localization of SepG and MyoB. We identify fourteen probable IQ-motifs (EF-hand protein binding sites) in the predicted SepG sequence. Two of the A. nidulans EF-hand proteins, myosin essential light chain (AnCdc4) and myosin regulatory light chain (MrlC), colocalize with SepG and MyoB at all stages of CAR formation and constriction. However, calmodulin (CamA) appears at septation sites only after the CAR has become fully compacted. When expression of sepG is downregulated, leaving MyoB as the sole IQ-motif protein in the pre-compaction CAR, both MrlC and AnCdc4 continue to associate with the forming CAR. When myoB expression is downregulated, leaving SepG as the sole IQ-motif protein in the CAR, AnCdc4 association with the forming CAR continues but MrlC fails to associate. This supports a model in which the IQ motifs of MyoB bind both MrlC and AnCdc4, while the IQ motifs of SepG bind only AnCdc4. Downregulation of either mrlC or Ancdc4 results in an aseptate phenotype, but has no effect on association of either SepG or MyoB with the CAR.

在这项研究中，我们报告了构巢曲霉中的sepG1突变位于基因 AN9463 中，该基因预计将编码 IQGAP 直向同源物。预计遗传损伤会导致 RasGAP-C 末端 (RGCT) 域的高度保守区域中 1737 残基蛋白的残基 1637 处发生 G 到 R 替换。当在限制温度下生长时，表达sepG ^G1637R ( sepG1) 等位基因是无间隔的，具有减少的菌落生长和异常形成的分生孢子梗。无菌条件可以通过删除 AN9463 或通过引入启动子下调其表达来复制。该突变不会阻止皮质收缩性肌动球蛋白环 (CAR) 在假定的分隔位点的组装，但环的紧密压实受损并且环无法收缩。GFP::SepG 野生型和sepG1等位基因的 GFP 标记产物在允许和限制温度下均定位于 CAR。myoB 的下调（编码构巢曲霉II 型肌球蛋白重链）不会阻止在分隔位点形成 SepG 环，但丝状肌动蛋白是 SepG 和 MyoB 的 CAR 定位所必需的。我们在预测的 SepG 序列中确定了十四个可能的 IQ 基序（EF 手蛋白结合位点）。所述的两个构巢曲霉EF-hand蛋白质，肌球蛋白必需轻链（AnCdc4）并于CAR形成和收缩所有阶段肌球蛋白调节轻链（MRLC），共定位与SEPG和MYOB。然而，钙调蛋白 (CamA) 仅在 CAR 完全压实后才会出现在分隔部位。当sepG 的表达下调时，MyoB 作为预压实 CAR 中唯一的 IQ 基序蛋白，MrlC 和 AnCdc4 继续与形成的 CAR 相关联。当myoB表达下调，使 SepG 成为 CAR 中唯一的 IQ 基序蛋白，AnCdc4 与形成 CAR 的关联继续，但 MrlC 未能关联。这支持一个模型，其中 MyoB 的 IQ 基序结合 MrlC 和 AnCdc4，而 SepG 的 IQ 基序仅结合 AnCdc4。mrlC或Ancdc4 的下调导致无菌表型，但对 SepG 或 MyoB 与 CAR 的关联没有影响。