期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：46
页码：E4929-E4935
DOI：10.1073/pnas.1411284111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceEnsuring the accuracy of protein interaction cascades is a challenge in many cellular processes. This challenge is faced by the guided entry of tail-anchored (TA) protein (GET) pathway, in which the targeting factor Get3 must sequentially interact with three effector proteins to deliver an essential class of TA proteins to the membrane. Using fluorescence probes that quantitatively interrogate individual Get3-effector interactions, we show here that Get3 adopts discrete conformational states in response to substrate and nucleotide binding; these conformational states allow Get3 to generate differential gradients of interaction energies with distinct effectors, thus driving its cyclic and ordered interaction cascade. These results also explain why multiple effector proteins are needed for TA targeting and uncover a previously unidentified mechanism for recycling Get3 from the membrane. Efficient and accurate localization of membrane proteins requires a complex cascade of interactions between protein machineries. This requirement is exemplified in the guided entry of tail-anchored (TA) protein (GET) pathway, where the central targeting factor Get3 must sequentially interact with three distinct binding partners to ensure the delivery of TA proteins to the endoplasmic reticulum (ER) membrane. To understand the molecular principles that provide the vectorial driving force of these interactions, we developed quantitative fluorescence assays to monitor Get3-effector interactions at each stage of targeting. We show that nucleotide and substrate generate differential gradients of interaction energies that drive the ordered interaction of Get3 with successive effectors. These data also provide more molecular details on how the targeting complex is captured and disassembled by the ER receptor and reveal a previously unidentified role for Get4/5 in recycling Get3 from the ER membrane at the end of the targeting reaction. These results provide general insights into how complex protein interaction cascades are coupled to energy inputs in biological systems.
关键词：protein targeting ; tail-anchored protein ; ATPase ; protein interaction cascades ; fluorescence
