Small, membrane-bound, alternatively spliced forms of ankyrin 1 associated with the sarcoplasmic reticulum of mammalian skeletal muscle.
Small, membrane-bound, alternatively spliced forms of ankyrin 1 associated with the sarcoplasmic reticulum of mammalian skeletal muscle.
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DOI：
10.1083/jcb.136.3.621
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发表时间：
1997-02-10
期刊：
影响因子：
--
通讯作者：
Bloch RJ
中科院分区：
文献类型：
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作者：
Zhou D;Birkenmeier CS;Williams MW;Sharp JJ;Barker JE;Bloch RJ
我们最近发现，红系Ankyrin基因ANK1在小鼠骨骼肌中表达异构体，其中一些与已知的ANK1异构体具有相同的COOH末端序列，但在其NH2末端有一个新的高度疏水的72个氨基酸片段。在这里，通过使用区域特异性多肽抗体，我们报告了小Anyrins在大鼠和兔骨骼肌中的存在，并证明了它们与肌浆网的选择性联系。在大鼠骨骼肌冰冻切片中，抗血影蛋白结合域的抗体(抗p65)仅与210-kD的ANK1反应并标记肌膜和细胞核，而针对小锚蛋白COOH端的抗体(抗p6)则与免疫印迹上20-26 kD的多肽反应并以网状图案装饰肌浆。纯合正常母细胞增殖性突变的小鼠(基因符号nb)缺乏210-kD的骨架蛋白，但在肌浆中含有正常水平的小骨架蛋白。在nb/nb骨骼肌中，抗p65标记在肌膜上缺失，而抗p6标记的分布与对照骨骼肌相同。在正常大鼠骨骼肌中，抗p6蛋白修饰Z线，如抗连接蛋白分布所定义的，也存在于M线，在那里它包裹着粗大的肌球蛋白细丝。用兔肌浆网分离的蛋白免疫印迹表明，小锚蛋白在这一组分中高度浓缩。当在转染的HEK 293细胞中表达时，如果存在NH2末端疏水结构域，则小Anyrins以类似于ER的网状模式分布，而如果不存在该结构域，则它们均匀分布在细胞质中。这些结果表明，小粘蛋白是肌浆网的完整膜蛋白。我们认为，与先前定位于肌膜并被认为是支持细胞骨架一部分的210-kD形式的ANK1不同，小的ANK1亚型可能通过将肌浆网连接到收缩装置来稳定肌浆网。
We have recently found that the erythroid ankyrin gene, Ank1, expresses isoforms in mouse skeletal muscle, several of which share COOH-terminal sequence with previously known Ank1 isoforms but have a novel, highly hydrophobic 72–amino acid segment at their NH2 termini. Here, through the use of domainspecific peptide antibodies, we report the presence of the small ankyrins in rat and rabbit skeletal muscle and demonstrate their selective association with the sarcoplasmic reticulum. In frozen sections of rat skeletal muscle, antibodies to the spectrin-binding domain (anti-p65) react only with a 210-kD Ank1 and label the sarcolemma and nuclei, while antibodies to the COOH terminus of the small ankyrin (anti-p6) react with peptides of 20 to 26 kD on immunoblots and decorate the myoplasm in a reticular pattern. Mice homozygous for the normoblastosis mutation (gene symbol nb) are deficient in the 210-kD ankyrin but contain normal levels of the small ankyrins in the myoplasm. In nb/nb skeletal muscle, anti-p65 label is absent from the sarcolemma, whereas anti-p6 label shows the same distribution as in control skeletal muscle. In normal skeletal muscle of the rat, anti-p6 decorates Z lines, as defined by antidesmin distribution, and is also present at M lines where it surrounds the thick myosin filaments. Immunoblots of the proteins isolated with rabbit sarcoplasmic reticulum indicate that the small ankyrins are highly enriched in this fraction. When expressed in transfected HEK 293 cells, the small ankyrins are distributed in a reticular pattern resembling the ER if the NH2-terminal hydrophobic domain is present, but they are uniformly distributed in the cytosol if this domain is absent. These results suggest that the small ankyrins are integral membrane proteins of the sarcoplasmic reticulum. We propose that, unlike the 210-kD form of Ank1, previously localized to the sarcolemma and believed to be a part of the supporting cytoskeleton, the small Ank1 isoforms may stabilize the sarcoplasmic reticulum by linking it to the contractile apparatus.