Biozentrum, University of Basel, Basel, Switzerland
Biozentrum, University of Basel, Basel, Switzerland.
Life Sci Alliance. 2022 Mar 21;5(7). doi: 10.26508/lsa.202101269. Print 2022 Mar.
Retrograde protein transport from the cell surface and endosomes to the TGN is essential for membrane homeostasis in general and for the recycling of mannose-6-phosphate receptors (MPRs) for sorting of lysosomal hydrolases in particular. We used a nanobody-based sulfation tool to more directly determine transport kinetics from the plasma membrane to the TGN for the cation-dependent MPR (CDMPR) with and without rapid or gradual inactivation of candidate machinery proteins. Although knockdown of retromer (Vps26), epsinR, or Rab9a reduced CDMPR arrival to the TGN, no effect was observed upon silencing of TIP47. Strikingly, when retrograde transport was analyzed by rapamycin-induced rapid depletion (knocksideways) or long-term depletion by knockdown of the clathrin adaptor AP-1 or of the GGA machinery, distinct phenotypes in sulfation kinetics were observed, suggesting a potential role of GGA adaptors in retrograde and anterograde transport. Our study illustrates the usefulness of derivatized, sulfation-competent nanobodies, reveals novel insights into CDMPR trafficking biology, and further outlines that the selection of machinery inactivation is critical for phenotype analysis.
从细胞表面和内体到 TGN 的逆行蛋白转运对于膜稳态是必不可少的，特别是对于甘露糖-6-磷酸受体 (MPR) 的循环利用，以便对溶酶体水解酶进行分拣。我们使用基于纳米抗体的硫酸化工具，更直接地确定阳离子依赖性 MPR（CDMPR）从质膜到 TGN 的转运动力学，同时快速或逐渐失活候选机器蛋白。尽管敲低 retromer（Vps26）、epsinR 或 Rab9a 会减少 CDMPR 到达 TGN，但沉默 TIP47 时没有观察到影响。引人注目的是，当通过雷帕霉素诱导的快速耗竭（knocksideways）或通过敲低网格蛋白衔接蛋白 AP-1 或 GGA 机器进行长期耗竭来分析逆行转运时，硫酸化动力学观察到不同的表型，这表明 GGA 衔接蛋白在逆行和顺行转运中可能发挥作用。我们的研究说明了衍生的、硫酸化能力的纳米抗体的有用性，揭示了 CDMPR 运输生物学的新见解，并进一步表明机器失活的选择对于表型分析至关重要。
