Blackbourn H D, Jackson A P
Biochemistry Department, University of Cambridge, UK.
J Cell Sci. 1996 Apr;109 ( Pt 4):777-86. doi: 10.1242/jcs.109.4.777.
Clathrin-coated vesicles were isolated from soybean (Glycine max L.) cells in suspension culture and their purity was assessed using SDS-PAGE, peptide sequencing and electron microscopy. Antibodies raised to these coated vesicles were used to immunoscreen a soybean cDNA library in lambda gt11 and isolate a partial clone of the clathrin heavy chain (HC) gene. Full-length cDNA for soybean clathrin HC was deduced by 5' and 3' cDNA amplification. The cDNA encodes an amino acid sequence of 1,700 residues, which is slightly larger than rat clathrin HC and may account for the reduced mobility of plant clathrin on SDS-PAGE. Insertion of these extra residues is largely confined to the amino and carboxy termini. Other domains within the heavy chain arms, including those implicated in light chain binding and trimerisation, are relatively well conserved between eukaryotes. A computer algorithm to determine alpha-helical coiled-coil structures reveals that only one domain, aligning to residues 1,460-1,489 in rat clathrin HC, has a high probability for coiled-coil structure in all five eukaryotic clathrin HC sequences. This provides further evidence that the interaction between clathrin heavy and light chains is mediated by three bundles of coiled-coils near to the carboxy terminus. In analysing the role of plant clathrin in endocytotic trafficking, as against trafficking from the Golgi apparatus to the vacuole, our attention was focused on membrane recycling in tip-growing pollen tubes. These rapidly growing cells are highly secretory and require a high level of plasma membrane recycling to maintain the tube tip architecture. Monoclonal antibodies to plant clathrin HC confirmed that coated vesicles are relatively abundant in tip-growing pollen tubes of Lilium longiflorum. This analysis also demonstrated that a high proportion of the clathrin present is in an assembled state, suggesting a highly dynamic trafficking pathway. Immunofluorescence analysis of pollen tubes revealed that clathrin localises to the plasma membrane at the apex of the pollen tube tip, which is consistent with high levels of clathrin-mediated membrane recycling. The use of these reagents in conjunction with tip-growing pollen tubes has created a unique opportunity to examine the basis for constitutive endocytosis, so that the more complex question of receptor-mediated pathways in plants can also be assessed.
从悬浮培养的大豆（Glycine max L.）细胞中分离出网格蛋白包被囊泡，并使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、肽测序和电子显微镜对其纯度进行评估。针对这些包被囊泡产生的抗体用于对λgt11载体中的大豆cDNA文库进行免疫筛选，并分离出网格蛋白重链（HC）基因的部分克隆。通过5'和3' cDNA扩增推导得到大豆网格蛋白HC的全长cDNA。该cDNA编码一个1700个残基的氨基酸序列，略大于大鼠网格蛋白HC，这可能解释了植物网格蛋白在SDS-PAGE上迁移率降低的原因。这些额外残基的插入主要局限于氨基和羧基末端。重链臂内的其他结构域，包括那些与轻链结合和三聚化有关的结构域，在真核生物之间相对保守。一种用于确定α-螺旋卷曲螺旋结构的计算机算法显示，在所有五个真核生物网格蛋白HC序列中，只有一个与大鼠网格蛋白HC中1460 - 1489位残基对齐的结构域具有形成卷曲螺旋结构的高概率。这进一步证明了网格蛋白重链和轻链之间的相互作用是由靠近羧基末端的三束卷曲螺旋介导的。在分析植物网格蛋白在内吞运输中的作用时，与从高尔基体到液泡的运输相对比，我们的注意力集中在顶端生长的花粉管中的膜循环上。这些快速生长的细胞具有高度分泌性，需要高水平的质膜循环来维持管尖结构。针对植物网格蛋白HC的单克隆抗体证实，在长花百合顶端生长的花粉管中，包被囊泡相对丰富。该分析还表明，存在的网格蛋白中有很大一部分处于组装状态，这表明存在高度动态的运输途径。对花粉管的免疫荧光分析显示，网格蛋白定位于花粉管顶端的质膜上，这与高水平的网格蛋白介导的膜循环一致。将这些试剂与顶端生长的花粉管结合使用，为研究组成型内吞作用的基础创造了独特的机会，从而也可以评估植物中受体介导途径这个更复杂的问题。
