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Proprotein convertase PC3 is not a transmembrane protein

机译：前蛋白转化酶PC3不是跨膜蛋白

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Proprotein convertase PC3 (also known as PC1) is an endopeptidase involved in proteolytic processing of peptide hormone precursors in granules of the regulated secretory pathway of endocrine cells. Lacking any extended hydrophobic segments, PC3 was considered to be a secretory protein only peripherally attached to the granule membrane. Recently, evidence has been presented that PC3 is a transmembrane protein with a 115-residue cytoplasmic domain and a membrane-spanning segment containing eight charged amino acids [Arnaoutova, I., et al. (2003) Biochemistry 42, 10445-10455]. Here, we analyzed the membrane topology of PO and of a PO construct containing a conventional transmembrane segment of 19 leucines. Alkaline extraction was performed to assess membrane integration. Exposure to the cytosol or to the ER lumen was tested by addition of C-terminal tags for phosphorylation or glycosylation, respectively. Protease sensitivity was assayed in permeabilized cells. The results show that the C-terminus of PC3 is translocated across the endoplasmic reticulum membrane. Furthermore, the proposed transmembrane segment of PC3 and a similar one of carboxypeptidase E did not stop polypeptide translocation when inserted into a stop-transfer tester construct. PC3 is thus not a transmembrane protein. These results have implications for the mechanism of granule sorting of PO as well as for the topology of PC2 and carboxypeptidase E, which have been reported to span the lipid membrane by homologous charged sequences.
机译：前蛋白转化酶PC3（也称为PC1）是一种内肽酶，参与内分泌细胞调节分泌途径颗粒中肽激素前体的蛋白水解加工。缺少任何延伸的疏水性片段，PC3被认为是仅在外围附着在颗粒膜上的一种分泌蛋白。最近，已经有证据表明PC3是具有115个残基的胞质结构域和跨膜段的跨膜蛋白，所述跨膜蛋白包含八个带电荷的氨基酸[Arnaoutova，I。，等人。 （2003）Biochemistry 42，10445-10455]。在这里，我们分析了PO和含有19个亮氨酸的常规跨膜片段的PO构建体的膜拓扑。进行碱性提取以评估膜整合。通过分别添加C末端标签的磷酸化或糖基化来测试暴露于细胞质或ER内腔。在透化细胞中测定蛋白酶敏感性。结果表明，PC3的C末端跨内质网膜移位。此外，拟议的PC3跨膜片段和类似的羧肽酶E插入终止转移测试仪构建物中时，不会阻止多肽移位。因此，PC3不是跨膜蛋白。这些结果对PO的颗粒分选机制以及PC2和羧肽酶E的拓扑结构都有影响，据报道，它们通过同源带电序列跨越脂质膜。

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