Yang Xi, Doray Balraj, Venkatarangan Varsha, Jennings Benjamin C, Henn Danielle, Liang Jiaxuan, Zhao Haikun, Zhang Weichao, Zhang Bokai, Yu Linchen, Chen Liang, Kornfeld Stuart, Li Ming
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
Current address: Department of Biological Sciences, Knoebel Institute for Healthy Aging, University of Denver, Denver, CO 80208, USA.
bioRxiv. 2024 Dec 5:2024.12.05.627003. doi: 10.1101/2024.12.05.627003.
In vertebrates, newly synthesized lysosomal enzymes traffick to lysosomes through the mannose-6-phosphate (M6P) pathway. The Golgi membrane protein TMEM251 was recently discovered to regulate lysosome biogenesis by controlling the level of GlcNAc-1-phosphotransferase (GNPT). However, its precise function remained unclear. In this study, we demonstrate that TMEM251 is a two-transmembrane protein indispensable for GNPT stability, cleavage by Site-1-Protease (S1P), and enzymatic activity. We reconcile conflicting models by showing that TMEM251 enhances GNPT cleavage and prevents its mislocalization to lysosomes for degradation. We further establish that TMEM251 achieves this by interacting with GOLPH3 and retromer complexes to anchor the TMEM251-GNPT complex at the Golgi. Alanine mutagenesis identified FXXR motif in TMEM251's N-tail for GOLPH3 binding. Together, our findings uncover TMEM251's multi-faceted role in stabilizing GNPT, retaining it at the Golgi, and ensuring the fidelity of the M6P pathway, thereby providing insights into its molecular function.
在脊椎动物中，新合成的溶酶体酶通过甘露糖-6-磷酸（M6P）途径运输到溶酶体。高尔基体膜蛋白TMEM251最近被发现通过控制N-乙酰葡糖胺-1-磷酸转移酶（GNPT）的水平来调节溶酶体生物发生。然而，其确切功能仍不清楚。在本研究中，我们证明TMEM251是一种双跨膜蛋白，对GNPT的稳定性、被位点1蛋白酶（S1P）切割以及酶活性不可或缺。我们通过表明TMEM251增强GNPT切割并防止其错误定位到溶酶体进行降解，调和了相互矛盾的模型。我们进一步确定TMEM251通过与GOLPH3和retromer复合物相互作用，将TMEM251-GNPT复合物锚定在高尔基体上来实现这一点。丙氨酸诱变确定了TMEM251 N端尾巴中用于与GOLPH3结合的FXXR基序。总之，我们的发现揭示了TMEM251在稳定GNPT、将其保留在高尔基体以及确保M6P途径的保真度方面的多方面作用，从而为其分子功能提供了见解。
