LITAF: coupling ubiquitination to transport at the endosome

细胞对环境的反应是由细胞表面表达的数百种不同的膜蛋白决定的。细胞必须不断取样和修饰这种表面膜蛋白的补体，去除由于环境变化而在表面不再需要的蛋白质，或者替换因日常压力而受损的蛋白质。执行这一重要“质量控制”的过程是内吞作用，即表面膜蛋白在囊泡内内化（“内吞”），到达细胞内腔室，即内核体。从那里，不再需要的膜蛋白被包装到核内体的内部囊泡中，然后产生的结构（多囊体）被运送到降解室（溶酶体）。另一种方法是，将被重新利用的表面膜蛋白包装在核内体的膜小管中，然后返回细胞表面。内体途径的每一步都涉及到为不同目的地选择货物膜蛋白的机制，以及使膜变形以产生运输中间体（即囊泡和小管）的机制。总之，内体途径的特点是具有丰富的高曲率膜，这些膜可以进行转运反应。几乎所有细胞蛋白的功能都受到翻译后修饰的调节。其中一个关键的修饰是泛素化（小蛋白泛素或多泛素链的共价附着）。泛素化是广泛存在的，细胞表达1000个泛素化酶（泛素连接酶），但在内吞过程中尤其重要，它执行几个重要任务。首先，溶酶体降解的靶膜蛋白被泛素标记，泛素被内体泛素受体识别，该受体选择这些货物并参与膜变形蛋白来执行每个运输步骤。其次，这些内体泛素受体本身可以被泛素化，导致它们的自我抑制，因此它们被关闭。第三，许多膜变形蛋白也被泛素化，通常导致它们快速和可逆的失活。它们的协同泛素化确保了内体途径内的转运反应是渐进和有效的。许多泛素化事件是由一个称为rsp5的泛素连接酶家族驱动的，其中ITCH是一个值得注意的成员。这些连接酶遍布整个细胞，因此它们必须受到“接头”蛋白的密切控制，以确保它们在正确的时间和地点泛素化正确的靶标。我们提出一种称为LITAF的蛋白质是激活内体途径内高曲率膜上的ITCH（和其他Rsp5连接酶）的关键接头，从而促进支持内体途径正常功能的泛素化事件的范围。我们的假设基于我们的发现：i) LITAF本身可以支持膜曲率，并且对内体运输很重要；ii) LITAF激活瘙痒；iii) LITAF与内体泛素受体结合；iv) LITAF和ITCH的缺失会在内体途径中产生类似的膜曲率缺陷。我们的建议有两个目的。首先，我们试图了解LITAF如何激活瘙痒，因此作为一个真正的瘙痒适配器，具有激活高曲率膜上的瘙痒的潜力。其次，我们将确定LITAF促进瘙痒依赖性泛素化的蛋白质。我们相信内体泛素受体可能是这类底物的关键类别，我们将对此进行测试。我们还将进行无偏见筛选，以寻找其他LITAF客户，寻找促进内体运输步骤的重要成分。对于其中的一小部分，我们将测试它们泛素化的影响。

How cells respond to their environment is determined by hundreds of different membrane proteins that are expressed on the cell surface. Cells must constantly sample and modify this complement of surface membrane proteins, to remove proteins that are no longer required at the surface due to a changed environment, or to replace proteins which become damaged by day-to-day stresses. The process that performs this important 'quality control' is endocytosis, whereby surface membrane proteins are internalised ('endocytosed') within vesicles to reach an intracellular compartment, the endosome. From there, membrane proteins that are no longer required are packaged into internal vesicles within the endosome, and the resulting structure (the multivesicular body) is then transported towards a degradative compartment (the lysosome). Alternatively, surface membrane proteins that are to be re-used are packaged at the endosome into membrane tubules and return to the cell surface. Each step of the endosomal pathway involves machinery that selects cargo membrane proteins for different destinations, coupled to machinery that deforms the membrane to generate transport intermediates (i.e. vesicles and tubules). In summary, the endosomal pathway is characterised by an abundance of high-curvature membranes that enact transport reactions. The function of virtually all cellular proteins is regulated by post-translational modifications. One key such modification is ubiquitination (the covalent attachment of the small protein ubiquitin, or polyubiquitin chains). Ubiquitination is widespread, with cells expressing >1000 ubiquitinating enzymes (ubiquitin ligases), but is particularly important during endocytosis, where it performs several important tasks. First, membrane proteins that are targeted for lysosomal degradation are tagged by ubiquitin, which is recognised by endosomal ubiquitin receptors that select such cargo and engage membrane-deforming proteins to enact each transport step. Second, these endosomal ubiquitin receptors can themselves be ubiquitinated, resulting in their auto-inhibition and thus they are switched off. Third, many membrane-deforming proteins are also ubiquitinated, often resulting in their rapid and reversible inactivation. Their coordinated ubiquitination ensures that transport reactions within the endosomal pathway are processive and efficient.Many of these ubiquitination events are driven by a family of ubiquitin ligases called Rsp5s, of which ITCH is a notable member. These ligases are located throughout the cell, so they must be closely controlled by 'adaptor' proteins to ensure that they ubiquitinate the correct targets at the right time and place. We propose that a protein called LITAF is the crucial adaptor that activates ITCH (and other Rsp5 ligases) upon the high-curvature membranes within the endosomal pathway and hence promotes the range of ubiquitination events that underpin the proper functioning of the endosomal pathway. We base our hypothesis on our findings: i) LITAF can itself support membrane curvature and is important for endosomal transport; ii) LITAF activates ITCH; iii) LITAF binds to endosomal ubiquitin receptors; iv) Depletion of LITAF and ITCH generate similar defects in membrane curvature within the endosomal pathway.Our proposal has two aims. First, we seek to understand how LITAF activates ITCH, and hence behaves as a bona fide ITCH adaptor with the potential to activate ITCH on high-curvature membranes. Second, we will identify the proteins for which LITAF promotes ITCH-dependent ubiquitination. We believe that endosomal ubiquitin receptors may be a key class of such substrates, and we will test this. We will also perform a non-biased screen to look for other LITAF clients, looking out for important components that facilitate endosomal transport steps. For a small subset of these we will test the impact of their ubiquitination.