GOLPH3 and GOLPH3L maintain Golgi localization of LYSET and a functional mannose 6-phosphate transport pathway

Glycosylation, which plays an important role in modifying lipids and sorting of proteins, is regulated by asymmetric intra-Golgi distribution and SPPL3-mediated cleavage of Golgi enzymes. We found that cells lacking LYSET/TMEM251, a retention factor for Golgi N-acetylglucosamine-1-phosphotransferase (GNPT), display SPPL3-dependent hypersecretion of the Golgi membrane protein B4GALT5. We demonstrate that in wild-type cells B4GALT5 is tagged with mannose 6-phosphate (M6P), a sorting tag typical of soluble lysosomal hydrolases Hence, M6P-tagging of B4GALT5 may represent a novel degradative lysosomal pathway. We also observed B4GALT5 hypersecretion and prominent destabilization of LYSET-GNPT complexes, impaired M6P-tagging and disturbed maturation and trafficking of lysosomal enzymes in multiple human cell lines lacking the COPI adaptors GOLPH3 and GOLPH3L. Mechanistically, we identified LYSET as a novel, atypical client of GOLPH3/GOLPH3L. Thus, by ensuring the cis-Golgi localization of the LYSET-GNPT complex and maintaining its Golgi polarity, GOLPH3/GOLPH3L is essential for the integrity of the M6P-tagging machinery and homeostasis of lysosomes.

糖基化（Glycosylation）在脂质修饰与蛋白质分选过程中发挥关键作用，其调控依赖于高尔基体内部的不对称分布，以及SPPL3介导的高尔基体酶类剪切。我们发现，缺失LYSET/TMEM251的细胞——该蛋白是高尔基体N-乙酰葡糖胺-1-磷酸转移酶（GNPT）的驻留因子——会呈现出SPPL3依赖性的高尔基体膜蛋白B4GALT5过度分泌表型。我们证实，在野生型细胞中，B4GALT5会被修饰上甘露糖6-磷酸（mannose 6-phosphate, M6P），这是可溶性溶酶体水解酶典型的分选标签；因此，B4GALT5的M6P修饰或许代表了一条全新的溶酶体降解通路。我们还在多种缺失COPI衔接蛋白（COPI adaptors）GOLPH3与GOLPH3L的人类细胞系中，观察到B4GALT5过度分泌、LYSET-GNPT复合物稳定性显著下降、M6P修饰受损，以及溶酶体酶的成熟与转运过程紊乱等异常表型。从分子机制层面，我们鉴定出LYSET是GOLPH3/GOLPH3L的新型非典型客户蛋白。综上，GOLPH3/GOLPH3L通过维持LYSET-GNPT复合物在顺式高尔基体（cis-Golgi）的定位，并维系高尔基体的极性，对于M6P修饰系统的完整性与溶酶体稳态至关重要。