Rodriguez-Polanco Wilmer R, Norris Anne, Velasco Agustin B, Gleason Adenrele M, Grant Barth D
bioRxiv. 2023 Feb 28:2023.02.27.530328. doi: 10.1101/2023.02.27.530328.
After endocytosis, many plasma membrane components are recycled via narrow-diameter membrane tubules that emerge from early endosomes to form recycling endosomes, eventually leading to their return to the plasma membrane. We previously showed that the F-BAR and SH3 domain Syndapin/PACSIN-family protein SDPN-1 is required for basolateral endocytic recycling in the intestine. Here we sought to determine the significance of a predicted interaction between the SDPN-1 SH3 domain and a target sequence in PXF-1/PDZ-GEF1/RAPGEF2, a known exchange factor for Rap-GTPases. We found that endogenous mutations we engineered into the SDPN-1 SH3 domain, or its binding site in the PXF-1 protein, interfere with recycling , as does loss of the PXF-1 target RAP-1. Rap-GTPases have been shown in several contexts to negatively regulate RhoA activity. Our results show that RHO-1/RhoA is enriched on SDPN-1 and RAP-1 positive endosomes in the intestine, and loss of SDPN-1 or RAP-1 elevates RHO-1(GTP) levels on intestinal endosomes. Furthermore, we found that depletion of RHO-1 suppressed mutant recycling defects, indicating that control of RHO-1 activity is a key mechanism by which SDPN-1 acts to promote endocytic recycling. RHO-1/RhoA is well-known for controlling actomyosin contraction cycles, although little is known of non-muscle myosin II on endosomes. Our analysis found that non-muscle myosin II is enriched on SDPN-1 positive endosomes, with two non-muscle myosin II heavy chain isoforms acting in apparent opposition. Depletion of inhibited recycling like mutants, while depletion of suppressed mutant recycling defects, indicating actomyosin contractility in controlling recycling endosome function.
内吞作用后，许多质膜成分通过从早期内体伸出的窄径膜小管进行回收，形成回收内体，最终导致它们回到质膜。我们之前表明，F-BAR和SH3结构域的Syndapin/PACSIN家族蛋白SDPN-1是肠道基底外侧内吞回收所必需的。在这里，我们试图确定SDPN-1 SH3结构域与PXF-1/PDZ-GEF1/RAPGEF2（一种已知的Rap小GTP酶交换因子）中的靶序列之间预测相互作用的意义。我们发现，我们在内源性SDPN-1 SH3结构域或其在PXF-1蛋白中的结合位点引入的突变会干扰回收，PXF-1的靶标RAP-1缺失也会如此。在多种情况下，Rap小GTP酶已被证明可负向调节RhoA活性。我们的结果表明，RHO-1/RhoA在肠道中SDPN-1和RAP-1阳性的内体上富集，SDPN-1或RAP-1的缺失会提高肠道内体上RHO-1（GTP）的水平。此外，我们发现RHO-1的缺失抑制了突变体的回收缺陷，表明控制RHO-1活性是SDPN-1促进内吞回收作用的关键机制。RHO-1/RhoA以控制肌动球蛋白收缩周期而闻名，尽管对内体上的非肌肉肌球蛋白II了解甚少。我们的分析发现，非肌肉肌球蛋白II在SDPN-1阳性内体上富集，两种非肌肉肌球蛋白II重链异构体的作用明显相反。缺失会像突变体一样抑制回收，而缺失则抑制突变体的回收缺陷，表明肌动球蛋白收缩性在控制回收内体功能中起作用。
