Characterization of novel interactors of Mucolipin-1

粘脂蛋白-1（MCOLN 1）的功能丧失突变导致IV型粘脂沉积症（MLIV），这是一种以严重的智力和精神发育迟滞为特征的溶酶体储存障碍。MCOLN 1是一种溶酶体离子通道，属于瞬时受体电位（TRP）超家族。为了更好地了解MCOLN 1的细胞功能，进行了分裂泛素酵母双杂交筛选，目的是揭示新的MCOLN 1相互作用子。筛选确定了溶酶体相关蛋白跨膜（LAPTM）家族的两个成员作为MCOLN 1的新型相互作用伴侣。通过免疫共沉淀和酵母双杂交试验证实了MCOLN 1和LAPTM之间的结合。此外，MCOLN 1和LAPTM广泛共定位于晚期内体-溶酶体。LAPTM 4 b的过表达引起溶酶体的增大和有缺陷的溶酶体降解，表明LAPTM 4 b对于适当的溶酶体功能是重要的。有趣的是，由LAPTM 4 b诱导的溶酶体肿胀被MCOLN 1的表达所拯救，这表明两种蛋白质之间存在功能性联系。最后，内源性LAPTM的siRNA耗竭诱导的同心多层结构和电子致密夹杂物的积累，非常类似于MLIV细胞中发现的结构。总的来说，我们的数据提供了新的见解MCOLN 1功能的分子机制，并提出了潜在的作用LAPTM MLIV发病机制。进一步的研究将试图确定由LAPTM转运的生理底物以及发现调节LAPTM活性的化合物。这些信息将进一步提高我们对MLIV的理解，并可能导致识别新的临床干预点。

Loss-of-function mutations in mucolipin-1 (MCOLN1) result in Mucolipidosis type IV (MLIV), a lysosomal storage disorder characterized by severe mental and psychomotor retardation. MCOLN1 is a lysosomal ion channel that belongs to the transient receptor potential (TRP) superfamily. To better understand the cellular function of MCOLN1, a split-ubiquitin yeast two-hybrid screen was performed with the purpose of revealing new MCOLN1 interactors. The screen identified two members of the lysosome-associated protein transmembrane (LAPTM) family as novel interaction partners of MCOLN1. The binding between MCOLN1 and LAPTMs was confirmed by co-immunoprecipitation and yeast two-hybrid assays. In addition, MCOLN1 and LAPTMs extensively co-localize at late endosomes-lysosomes. Over-expression of LAPTM4b caused enlargement of lysosomes and defective lysosomal degradation, indicating that LAPTMs are important for proper lysosomal function. Interestingly, lysosomal swelling induced by LAPTM4b was rescued by expression of MCOLN1, suggesting a functional connection between the two proteins. Finally, depletion of endogenous LAPTMs by siRNA induced accumulation of concentric multi-lamellar structures and electron-dense inclusions that closely resemble the structures found in MLIV cells. Overall, our data provide new insight into the molecular mechanisms of MCOLN1 function and suggest a potential role of LAPTMs in MLIV pathogenesis. Further studies will try to identify the physiological substrates transported by LAPTMs as well as discover compounds that modulate LAPTM activity. This information will further improve our understanding of MLIV and may lead to the identification of novel clinical intervention points.