Biotechnology Research Center, University of Tokyo, Tokyo 113-8657, Japan.
J Biol Chem. 2011 Feb 25;286(8):6175-83. doi: 10.1074/jbc.M110.184929. Epub 2010 Dec 9.
Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono- or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles.
硼素稳态对植物很重要，因为硼虽为必需元素，但过量则有毒。在高硼条件下，拟南芥硼酸转运蛋白 BOR1 通过内吞途径从质膜（PM）转运到液泡进行降解，以避免过量硼素的运输。在这里，我们表明硼诱导的泛素化对于 BOR1 的液泡分选是必需的。我们发现，BOR1 中的赖氨酸 590 突变为丙氨酸（K590A）会阻止降解。硼处理后几分钟内，BOR1 就会被单泛素化或双泛素化，而 K590A 突变体则不会。K590A 突变消除了 BOR1 的液泡运输，但对其向 PM 内域的极性定位似乎没有明显影响。此外，布雷菲德菌素 A 和渥曼青霉素处理表明 Lys-590 对于 BOR1 从早期内体区室到多泡体的易位是必需的。我们的结果表明，硼诱导的 BOR1 泛素化对于从 PM 进行内吞作用不是必需的，但对于将内化的 BOR1 分选到多泡体以随后在液泡中降解是至关重要的。
