Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA 22908.
Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, Charlottesville, VA 22908
Proc Natl Acad Sci U S A. 2017 Nov 7;114(45):12039-12044. doi: 10.1073/pnas.1709060114. Epub 2017 Oct 23.
Membrane contact sites (MCS) are zones of contact between the membranes of two organelles. At MCS, specific proteins tether the organelles in close proximity and mediate the nonvesicular trafficking of lipids and ions between the two organelles. The endoplasmic reticulum (ER) integral membrane protein VAP is a common component of MCS involved in both tethering and lipid transfer by binding directly to proteins containing a FFAT [two phenylalanines (FF) in an acidic tract (AT)] motif. In addition to maintaining cell homeostasis, MCS formation recently emerged as a mechanism by which intracellular pathogens hijack cellular resources and establish their replication niche. Here, we investigated the mechanism by which the containing vacuole, termed the inclusion, establishes direct contact with the ER. We show that the protein IncV, which is inserted into the inclusion membrane, displays one canonical and one noncanonical FFAT motif that cooperatively mediated the interaction of IncV with VAP. IncV overexpression was sufficient to bring the ER in close proximity of IncV-containing membranes. Although IncV deletion partially decreased VAP association with the inclusion, it did not suppress the formation of ER-inclusion MCS, suggesting the existence of redundant mechanisms in MCS formation. We propose a model in which IncV acts as one of the primary tethers that contribute to the formation of ER-inclusion MCS. Our results highlight a previously unidentified mechanism of bacterial pathogenesis and support the notion that cooperation of two FFAT motifs may be a common feature of VAP-mediated MCS formation. -host cell interaction therefore constitutes a unique system to decipher the molecular mechanisms underlying MCS formation.
膜接触位点（MCS）是两个细胞器的膜之间的接触区域。在 MCS 中，特定的蛋白质将细胞器系绳在近距离，并介导两个细胞器之间的脂质和离子的非囊泡运输。内质网（ER）的整合膜蛋白 VAP 是 MCS 的常见组成部分，通过直接与含有 FFAT [酸性结构域（AT）中的两个苯丙氨酸（FF）]基序的蛋白质结合，参与系绳和脂质转移。除了维持细胞内稳态外，MCS 的形成最近成为细胞内病原体劫持细胞资源并建立其复制生态位的一种机制。在这里，我们研究了包含体（称为包涵体）与 ER 建立直接接触的机制。我们表明，插入包涵体膜的 蛋白 IncV 显示出一个典型的和一个非典型的 FFAT 基序，共同介导 IncV 与 VAP 的相互作用。IncV 的过表达足以使 ER 接近含有 IncV 的膜。尽管 IncV 的缺失部分减少了 VAP 与包涵体的关联，但它并没有抑制 ER-包涵体 MCS 的形成，这表明 MCS 形成中存在冗余机制。我们提出了一个模型，其中 IncV 作为主要系绳之一，有助于 ER-包涵体 MCS 的形成。我们的结果强调了细菌发病机制的一个以前未被识别的机制，并支持了两个 FFAT 基序的合作可能是 VAP 介导的 MCS 形成的共同特征的观点。因此，宿主细胞相互作用构成了一个独特的系统，可以破译 MCS 形成的分子机制。
