Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is a four-membrane spanning ceramide interacting protein that regulates mTORC1 signaling. Here, we show that LAPTM4B is sorted into intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) and released in small extracellular vesicles (sEVs) into conditioned cell culture medium and human urine. Efficient sorting of LAPTM4B into ILV membranes depends on its third transmembrane domain containing a sphingolipid interaction motif (SLim). Unbiased lipidomic analysis reveals a strong enrichment of glycosphingolipids in sEVs secreted from LAPTM4B knockout cells and from cells expressing a SLim-deficient LAPTM4B mutant. The altered sphingolipid profile is accompanied by a distinct SLim-dependent co-modulation of ether lipid species. The changes in the lipid composition of sEVs derived from LAPTM4B knockout cells is reflected by an increased stability of membrane nanodomains of sEVs. These results identify LAPTM4B as a determinant of the glycosphingolipid profile and membrane properties of sEVs.

溶酶体相关蛋白跨膜4B（LAPTM4B）是一种跨膜的神经酰胺相互作用蛋白，调节mTORC1信号传导的四膜。在这里，我们显示，LAPTM4B被分类到多囊泡内体（MVE）的腔内囊泡（ILVs）中，并在小细胞外囊泡（sEVs）中释放到条件细胞培养基和人尿中。LAPTM4B是否能有效地分选到ILV膜中取决于它的第三个跨膜结构域，该结构域包含鞘脂相互作用基序（SLim）。无偏见的脂质组学分析显示，在从LAPTM4B敲除细胞和表达SLim缺失的LAPTM4B突变体的细胞分泌的sEV中，糖鞘脂的含量很高。改变的鞘脂谱伴随着醚脂质种类的独特的SLim依赖性共调节。衍生自LAPTM4B基因敲除细胞的sEV脂质组成的变化反映在sEV膜纳米域稳定性的提高上。这些结果确定了LAPTM4B是sEVs的糖鞘脂谱和膜特性的决定因素。