Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA.
PLoS One. 2013;8(1):e53366. doi: 10.1371/journal.pone.0053366. Epub 2013 Jan 24.
Flagellar assembly requires coordination between the assembly of axonemal proteins and the assembly of the flagellar membrane and membrane proteins. Fully grown steady-state Chlamydomonas flagella release flagellar vesicles from their tips and failure to resupply membrane should affect flagellar length. To study vesicle release, plasma and flagellar membrane surface proteins were vectorially pulse-labeled and flagella and vesicles were analyzed for biotinylated proteins. Based on the quantity of biotinylated proteins in purified vesicles, steady-state flagella appeared to shed a minimum of 16% of their surface membrane per hour, equivalent to a complete flagellar membrane being released every 6 hrs or less. Brefeldin-A destroyed Chlamydomonas Golgi, inhibited the secretory pathway, inhibited flagellar regeneration, and induced full-length flagella to disassemble within 6 hrs, consistent with flagellar disassembly being induced by a failure to resupply membrane. In contrast to membrane lipids, a pool of biotinylatable membrane proteins was identified that was sufficient to resupply flagella as they released vesicles for 6 hrs in the absence of protein synthesis and to support one and nearly two regenerations of flagella following amputation. These studies reveal the importance of the secretory pathway to assemble and maintain full-length flagella.
鞭毛组装需要轴丝蛋白的组装和鞭毛膜及膜蛋白的组装之间的协调。完全生长的稳定状态的衣藻鞭毛从其尖端释放鞭毛囊泡，如果不能补充膜，应该会影响鞭毛长度。为了研究囊泡的释放，对质膜和鞭毛膜表面蛋白进行了载体脉冲标记，并对鞭毛和囊泡进行了生物素化蛋白分析。根据纯化囊泡中生物素化蛋白的数量，稳态鞭毛每小时似乎至少丢失 16%的表面膜，相当于每 6 小时或更短时间就会释放完整的鞭毛膜。布雷菲德菌素 A 破坏了衣藻高尔基体，抑制了分泌途径，抑制了鞭毛再生，并在 6 小时内诱导全长鞭毛解体，这与不能补充膜会诱导鞭毛解体的观点一致。与膜脂不同，鉴定出了一个可生物素化的膜蛋白池，该池足以在没有蛋白质合成的情况下为囊泡的释放提供 6 小时的鞭毛，并在切除后支持一次和近两次的鞭毛再生。这些研究揭示了分泌途径对于组装和维持全长鞭毛的重要性。
