Premelanosome amyloid-like fibrils are composed of only Golgi-processed forms of Pmel17 that have been proteolytically processed in Endosomes
Premelanosome amyloid-like fibrils are composed of only Golgi-processed forms of Pmel17 that have been proteolytically processed in Endosomes
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DOI：
10.1074/jbc.m708007200
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发表时间：
2008-01-25
影响因子：
4.8
通讯作者：
Marks, Michael S.
中科院分区：
文献类型：
--
作者：
Harper, Dawn C.;Theos, Alexander C.;Marks, Michael S.
黑色素在称为黑素体的专门细胞器和黑素体前体内形成的管腔内原纤维中合成。原纤维由来自色素细胞特异性整合膜蛋白Pmel 17的蛋白水解片段组成。Pmel 17进入黑素体前体的细胞内途径和纤维状黑素体内Pmel 17衍生物的身份一直是一个争论的问题。我们在此表明，检测纤维状黑素体内Pmel 17的抗体仅识别前蛋白转化酶切割的管腔产物，而不识别与跨膜结构域连接的其余产物。此外，针对N和C末端的抗体仅检测存在于早期生物合成隔室中的Pmel 17同种型，其构成细胞裂解物中可检测的稳态Pmel 17的大部分，因为早期生物合成运输缓慢且通过原纤维形成快速消耗。使用一种抗体的管腔表位，被破坏后修改O-连接的寡糖，我们表明，所有后内质网Pmel 17亚型被高尔基体相关的寡糖转移酶的修改，只有加工形式有助于黑素体生物合成。这些数据表明，Pmel 17遵循一个单一的生物合成途径，从内质网通过高尔基体复合体和内体的黑素体，只有片段包含先前描述的功能管腔决定簇内的原纤维。这些数据对纤维形成的位点和机制具有重要意义。
Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. Weshow here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.