Mechanisms of endocytic recycling

摘要 内吞循环对于广泛的生理活动是至关重要的，包括营养摄取、细胞运动 以及极性、细胞内信号和胞质分裂。我们一直在研究一种皮毛复合体，它形成了 在循环途径中运输囊泡，这涉及ACAP1(带有线圈的ArfGap、Ankyrin重复序列和PH 结构域类型1)作为内部组分，而网状蛋白作为外层涂层。最近，我们做了一个 不同寻常的发现，发现蛋白激酶Akt作为这种外套复合体的另一种成分。AKT 也具有直接弯曲细胞膜的能力，这一发现是史无前例的，因为目前还没有人知道 拥有这种能力。我们最近的发现也使我们通过这一涂层重建了囊泡的形成。 很复杂。总而言之，这些发现使我们提出了三个主要目标。首先，最终了解 蛋白质如何弯曲薄膜是通过高分辨率冷冻电子显微镜(EM)实现的 解决组装在膜上的蛋白质的结构的方法。因为这是的函数形式 关于外壳蛋白，我们将与一个在基于EM的方法方面拥有丰富专业知识的团队合作 从分子细节上阐明外壳因子如何组装成膜上的蛋白质晶格结构以实现 膜弯曲。第二，尽管以蛋白质为基础的囊泡运输机制 在调查中，基于脂质的机制被研究得少得多。解决了这一根本缺点， 我们最近研究了小泡重建系统，以确定小泡所需的脂酶。 由ACAP1包衣复合体形成。因此，我们将阐明囊泡形成的特定阶段 需要一种特殊的酶。此外，为了更全面地了解脂肪产品是如何 一种特定的酶起作用，我们将探索所产生的脂质的几何形状是否影响ACAP1囊泡 形成，以及特定的脂类几何形状是否促进ACAP1衣壳因子的弯曲能力 薄膜。第三，我们最近对重组的ACAP1囊泡进行了质谱分析 牵涉到许多ACAP1途径的货物。为了验证这一发现，我们将重点关注意想不到的货物 对于进一步的研究，因为确认它们使用ACAP1途径将提供特别令人信服的 支持我们的方法已经确定了ACAP1途径的真实货物。具体地说，我们将变异 ACAP1涂层复合体识别的这些意外货物中的序列，然后确认分拣 进入ACAP1途径被抑制。我们还将寻求对意外货物的统一解释 使用ACAP1途径，通过确定这种运输是否导致它们传递到不动足纲，这 是局部的细胞表面结构，集中了基质降解的关键因素，这是 细胞侵入组织。我们预计，这些研究的完成不仅将进一步推动 对细胞内循环的基本了解，也为生理作用提供了新的见解 通过ACAP1途径。

ABSTRACT Endocytic recycling is critical for a broad range of physiologic events, including nutrient uptake, cell motility and polarity, intracellular signaling, and cytokinesis. We have been studying a coat complex that forms transport vesicles in a recycling pathway, which involves ACAP1 (Arfgap with Coil-coil, Ankyrin repeat and PH domain type 1) acting as the inner component and clathrin as the outer coating. Recently, we have made a remarkable discovery, finding that the protein kinase Akt acts as another component of this coat complex. Akt also possesses a direct ability to bend membrane, a finding that is unprecedented, as no kinase is known to possess this capability. Our recent discovery has also led us to reconstitute vesicle formation by this coat complex. Altogether, these findings lead us to propose three major goals. First, an ultimate understanding of how a protein bends membrane is being achieved through a high-resolution cryo-electron microscopy (EM) approach that solves the structure of the protein assembled on membrane. As this is the functional form of coat proteins, we will collaborate with a group having demonstrated expertise in the EM-based approach to elucidate in molecular detail how coat factors assemble into a protein lattice structure on membrane to achieve membrane bending. Second, whereas protein-based mechanisms of vesicular transport are being intensely investigated, lipid-based mechanisms have been far less explored. Addressing this fundamental shortcoming, we have recently pursued the vesicle reconstitution system to identify lipid enzymes needed for vesicle formation by the ACAP1 coat complex. Thus, we will elucidate the specific stage of vesicle formation that requires a particular enzyme. Moreover, to achieve a more complete understanding of how the lipid product of a particular enzyme acts, we will explore whether the geometry of the produced lipid affects ACAP1 vesicle formation, and also whether a particular lipid geometry promotes the ability of the ACAP1 coat factors to bend membrane. Third, we have recently performed mass spectrometry on the reconstituted ACAP1 vesicles to implicate many cargoes of the ACAP1 pathway. To validate this finding, we will focus on unexpected cargoes for further scrutiny, as confirmation that they use the ACAP1 pathway will provide particularly compelling support that our approach has identified true cargoes of the ACAP1 pathway. Specifically, we will mutate the sequence in these unexpected cargoes recognized by the ACAP1 coat complex and then confirm that sorting into the ACAP1 pathway is inhibited. We will also pursue an unifying explanation for the unexpected cargoes using the ACAP1 pathway by determining whether this transport results in their delivery to invadopodia, which are localized cell-surface structures that concentrate key factors for matrix degradation, a process needed for cell invasion into tissue. We anticipate that the completion of these studies will not only advance a further fundamental understanding of endocytic recycling, but also shed new insights into the physiologic roles served by the ACAP1 pathway.