A model for melanosome biogenesis based on the purification and analysis of early melanosomes

Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.

本研究通过纯化早期黑素体（melanosome）并对分选至该细胞器的特异性蛋白进行表征，针对黑素体的生物发生与功能展开了系统研究。研究人员先对高色素化的人MNT1黑色素瘤细胞进行裂解，再通过蔗糖密度梯度离心完成初步分离，从而从中分离得到黑素体。经电子显微镜检测发现，低密度蔗糖组分中富集了I期与II期黑素体，随后通过自由流电泳对该组分进行进一步分离与纯化。尽管多巴色素互变异构酶（dopachrome tautomerase, DCT）以及部分酪氨酸酶（tyrosinase）蛋白可被分选至I期黑素体，但二者的催化活性仅在II期黑素体中可检测到。蛋白质免疫印迹（Western immunoblotting）检测结果显示，上述催化蛋白以及酪氨酸酶相关蛋白1（TYRP1）、MART1、GP100均在I期黑素体中被剪切并失活。蛋白水解剪切对于GP100在黑素体微环境中的重折叠至关重要；而无定形的I期黑素体后续重组织为纤维状、卵圆形且高度结构化的II期黑素体，这一过程可稳定黑素体酶的催化功能，进而启动黑色素生物合成。本研究结果有助于更深入地理解黑素体生物发生过程中的结构特征，并为色素沉着的生理调控机制提供了新的线索。