Faculty of Biology, University of Athens, Panepistimioupolis 15781, Athens, Greece.
Mol Microbiol. 2010 Jan;75(1):246-60. doi: 10.1111/j.1365-2958.2009.06997.x. Epub 2009 Dec 11.
In this work we unmask a novel downregulation mechanism of the uric acid/xanthine transporter UapA, the prototype member of the ubiquitous Nucleobase-Ascorbate Transporter family, directly related to its function. In the presence of substrates, UapA is endocytosed, sorted into the multivesicular body pathway and degraded in vacuoles. Substrate-induced endocytosis, unlike ammonium-induced turnover, is absolutely dependent on UapA activity and several lines of evidence showed that the signal for increased endocytosis is the actual translocation of substrates through the UapA protein. The use of several UapA functional mutants with altered kinetics and specificity has further shown that transport-dependent UapA endocytosis occurs through a mechanism, which senses subtle conformational changes associated with the transport cycle. We also show that distinct mechanisms of UapA endocytosis necessitate ubiquitination of a single Lys residue (K572) by HulA(Rsp5). Finally, we demonstrate that in the presence of substrates, non-functional UapA versions can be endocytosed in trans if expressed in the simultaneous presence of active UapA versions, even if the latter cannot be endocytosed themselves.
在这项工作中，我们揭示了尿酸/黄嘌呤转运蛋白 UapA 的一种新型下调机制，该蛋白是广泛存在的核苷酸盐转运体家族的原型成员，与该蛋白的功能直接相关。在底物存在的情况下，UapA 被内吞，分选到多泡体途径，并在液泡中降解。与铵盐诱导的周转率不同，底物诱导的内吞作用绝对依赖于 UapA 的活性，并且有几条证据表明，增加内吞作用的信号是底物实际通过 UapA 蛋白的转运。使用几种具有改变的动力学和特异性的 UapA 功能突变体进一步表明，依赖于转运的 UapA 内吞作用通过一种机制发生，该机制感知与转运循环相关的微妙构象变化。我们还表明，UapA 内吞作用的不同机制需要 HulA(Rsp5)对单个赖氨酸残基（K572）进行泛素化。最后，我们证明，在存在底物的情况下，如果在活性 UapA 变体的同时表达，即使后者本身不能被内吞，无功能的 UapA 变体也可以通过转染被内吞。
