Department of Plant Physiology, Umeå Plant Science Centre, Umeå University, Umeå, Sweden.
PLoS One. 2011;6(6):e21021. doi: 10.1371/journal.pone.0021021. Epub 2011 Jun 10.
The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells.
METHODOLOGY/PRINCIPAL FINDINGS: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited.
CONCLUSIONS/SIGNIFICANCE: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.
拟南芥 CAH1α 型碳酸酐酶是少数几种已知通过分泌途径靶向叶绿体的植物蛋白之一。CAH1 在几个残基上通过附着 N-糖基化进行翻译后修饰，从而产生具有复杂型糖基的成熟蛋白。通过这种非典型途径运输对某些叶绿体驻留蛋白有益的原因尚不清楚。因此，为了阐明糖基化在运输中的意义以及糖基化对蛋白质稳定性和功能的影响，我们在植物细胞中表达了 CAH1 的野生型和突变型的表位标记版本。
方法/主要发现：具有破坏糖基化位点的突变 CAH1 的瞬时表达表明，该蛋白含有四个或在某些情况下五个 N-糖基。虽然野生型蛋白通过分泌途径运输到叶绿体，但非糖基化的蛋白形成聚集体并与内质网伴侣 BiP 相关联，表明 CAH1 的糖基化有助于折叠和 ER 输出。使用半胱氨酸突变体，我们还评估了二硫键形成在 CAH1 折叠和稳定性中的作用。我们发现成熟蛋白中第 27 和 191 位的半胱氨酸之间的二硫键对于蛋白的正确折叠是必需的。使用质谱方法，我们能够测量 CAH1 蛋白的酶活性。在体内阻断蛋白 N-糖基化的情况下，CAH1 的活性完全被抑制。
结论/意义：我们首次表明，在高等植物中，从分泌途径到叶绿体的蛋白质折叠和运输中，翻译后修饰（如 N-糖基化和分子内二硫键形成）的重要性。这些翻译后修饰对于完全功能性天然蛋白的要求解释了替代途径到叶绿体的必要性。
