Translocation of Proteins Across the Endoplasmic Reticulum
Translocation of Proteins Across the Endoplasmic Reticulum
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蛋白质穿过内质网的易位
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发表时间：
1981
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通讯作者：
Peter Walter
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作者：
Peter Walter
狗胰腺的易位态微粒体膜囊泡可以选择性地结合新生的体外组装的合成分泌蛋白（牛泌乳素）的多体，但不能结合合成细胞质蛋白（兔球蛋白α链和β链）的多体。当微粒体囊泡被盐萃取时，这种选择性多体结合能力被破坏，但通过先前从微粒体囊泡的盐萃取物中纯化的11S蛋白（SRP，信号识别蛋白）得以恢复（Walter和Blobel， 1980）。Proc。Nat /。专科学校科学。U。美国。77:7112-7116)。srp依赖性多体识别和结合微粒体膜被证明是链易位的先决条件。用n -乙基马来酰亚胺修饰SRP，证实了其介导新生多体与微粒体囊泡结合的能力。同样，当亮氨酸类似物8-羟基亮氨酸被纳入新生的分泌多肽时，与微粒体膜的多体结合在很大程度上被破坏。本论文和前一篇论文的数据提供了确凿的实验证据，证明跨内质网膜的链式易位是一个受体介导的事件，从而排除了链式易位自发发生、不受蛋白质介导的说法。此外，我们这里的数据确凿地表明，导致易位并提供其特异性的初始事件是蛋白质-蛋白质（信号序列加上带有SRP的核糖体）而不是蛋白质-脂质（信号序列与1脂质双层）相互作用。在之前的文章(1)中，我们已经描述了信号识别蛋白（SRP）在小麦生殖细胞自由翻译系统中的作用，该系统由mRNA编程，用于兔珠蛋白（细胞质蛋白）或牛催乳素（分泌蛋白）。我们已经证明(1)SRP特异性地与单体核糖体结合，尽管其附着度相对较低（表观kD < 5 × 10-5 M），并且它选择性地与体外组装的合成分泌蛋白的多体结合，附着度高6000倍（表观kD < 8 × 10-9 M）（但不与合成球蛋白的多体结合）。这种6000倍的增强很可能是由于SRP对新生分泌多肽的信号序列的特异性识别。在本文中，我们描述了SRP在添加狗胰腺微粒体膜小泡的小麦生殖细胞自由翻译系统中的特异性作用。我们的数据表明，SRP介导了合成分泌蛋白的新生体外组装多聚体与微粒体膜的选择性结合，而不是那些合成细胞质蛋白的多聚体。《细胞生物学杂志》1981年11月91卷551-556©THE rockefeller University Press 0021-9525 /81 /11 /0551 /06 $1。材料与方法在前一篇文章(1)中描述了各种微体膜组分（RM, K-RM）的制备、SRP的提取和纯化、无细胞小麦胚芽翻译系统以及体外合成蛋白的定量。除另有说明外，所使用的SRP制剂为氨基戊基琼脂糖树脂的洗脱液。体外组装多聚体与微粒体膜结合的试验初生多聚体与微粒体膜结合的试验是间接的。我们测量了翻译系统中由于招募到膜结合多体和通过差速离心去除这些体外组装的粗糙微粒体（RM）而导致的mRNA损耗。每个时间点的初始孵育体积为75 dal。将小麦生殖细胞自由翻译系统和其他组分（见2006年3月7日刊登的leg551号图）混合在冰上。系统在26°C水浴中加热2分钟；然后加入mRNA，开始预孵育（26℃）。通过将混合物在冰水中冷却至0°C，在不同的预孵育时间后，平移被阻止。取出一个40-Al的试管（留下一个35 - al的对照试管），转移到离心管中，并在保持在4°C的Beckman离心机（Beckman Instruments, Inc., fullerton, Cal i f）中在A100-30转子中以20 PSI旋转。为了启动旋转，在10秒后将气压慢慢调高至20 PSI。然后将样品再离心90秒。刹车在滑行两分钟后失灵。从顶部小心地取出35-p1的部分。将该样品与35-p1对照瓶（未纺丝）在26°C下持续孵育，直到每个样品的总孵育时间达到90分钟。然后分别取25pl的tca沉淀并制备SDS-PAGE。随着预孵育时间的增加，催乳素mRNA数量的增加将被组装成膜结合的多体，因此，通过沉淀从易位混合物中去除。上清液部分的孵育预计会导致所有游离mRNA和参与游离多体的mRNA的翻译。在使用的条件下，在我们的翻译系统中生成催乳素前的量与加入到翻译中的mRNA的量成正比。因此，催乳素的最终量是离心步骤后上清液中留下的可翻译mRNA量的函数，从而是在任何给定的预孵育时间内未附着在微粒体膜上的mRNA量的测量。
Translocation-competent microsomal membrane vesicles of dog pancreas were shown to selectively bind nascent, in vitro assembled polysomes synthesizing secretory protein (bovine prolactin) but not those synthesizing cytoplasmic protein (alpha and beta chain of rabbit globin) . This selective polysome binding capacity was abol ished when the microsomal vesicles were salt -extracted but was restored by an 11S protein (SRP, Signal Recognit ion Protein) previously puri f ied from the salt extract of microsomal vesicles (Walter and Blobel , 1980 . Proc . Nat / . Acad . Sci . U . S. A . 77 :7112-7116) . SRP-dependent polysome recognit ion and binding to the microsomal membrane was shown to be a prerequisite for chain translocation . Modi f ication of SRP by N-ethyl maleimide abol ished its abi l ity to mediate nascent polysome binding to the microsomal vesicles . Likewise, polysome binding to the microsomal membrane was largely abol ished when 8-hydroxy leucine, a Leu analogue, was incorporated into nascent secretory polypeptides . The data in this and the preceding paper provide conclusive experimental evidence that chain translocation across the endoplasmic reticulum membrane is a receptor-mediated event and thus rule out proposals that chain translocation occurs spontaneously and without the mediation by proteins. Moreover, our data here demonstrate conclusively that the init ial events that lead to translocation and provide for its speci f icity are protein-protein (signal sequence plus ribosome with SRP) and not proteinl ipid (signal sequence with l ipid bi layer) interactions . In the preceding paper (1) we have described the effects of signal recognit ion protein (SRP) in a wheat germ cel l free translation system programmed with mRNA 's for either rabbit globin (cytoplasmic protein) or bovine prolactin (secretory protein) . We have demonstrated (1) that SRP binds speci f ical ly to monomeric ribosomes, albeit with relatively low aff inity (apparent kD < 5 x 10-5 M) , and that it binds selectively and with 6,000fold higher aff inity (apparent kD < 8 x 10-9 M) to in vitro assembled polysomes synthesizing secretory protein (but not to those synthesizing globin) . This 6,000fold enhancement most l ikely results from speci f ic recognit ion by SRP of the signal sequence of the nascent secretory polypeptide . In this paper we describe the speci f ic effects of SRP in a wheat germ cel l free translation system that was supplemented with dog pancreas microsomal membrane vesicles . Our data show that SRP mediates the selective binding to microsomal membranes of nascent, in vitro assembled polysomes synthesizing secretory protein but not of those synthesizing cytoplasmic protein . Polysome binding is abol ished i f SRP is modi f ied THE JOURNAL OF CELL BIOLOGY VOLUME 91 NOVEMBER 1981 551-556 ©The Rockefel ler University Press 0021-9525 /81 /11 /0551 /06 $1 .00 by N-ethyl maleimide (NEM) or i f the nascent secretory polypeptide is modi f ied by incorporation of a-hydroxy leucine, an analogue of Leu. MATERIALS AND METHODS The preparation of various microsomel membrane fractions (RM, K-RM) , the extraction and puri f ication of SRP, the cel l free wheat germ translation system, and the quantitation of in vitro synthesized protein were described in the preceding paper (1) . The SRP preparation used was the eluate oftheaminopentyl agarose resin, except when stated otherwise . Assay for Binding of In Vitro Assembled Polysomes to Microsomal Membranes The assay for nascent polysome binding to microsomal membranes was an indirect one. We measured the depletion of mRNA from the translation system result ing from recruitment into membrane-bound polysomes and removal of these in vitro assembled rough microsomes (RM) by di fferential centri fugation . The init ial incubation volume for each timepoint was 75 dal. The wheat germ cel l free translation system plus addit ional components (speci f ied in f igure leg551 on M rch 7, 2006 ww.jcb.org Doloaded rom ends) were mixed on ice . The system was al lowed to warm for 2 min in a 26°C water bath ; mRNA was then added and preincubation (at 26°C) was started . The translations were arrested after various preincubation times by cool ing the mixture to 0°C in ice-water . A 40-Al al iquot was withdrawn ( leaving behind a 35Al control al iquot) , transferred to a centri fuge tube, and spun at 20 PSI in the A100-30 rotor in a Beckman Airfuge kept at 4°C (Beckman Instruments, Inc. , Ful lerton, Cal i f) . To start the spin, the air pressure was slowly turned up to reach 20 PSI after 10 s . The sample was then centri fuged for an addit ional 90 s. The brake engaged after a 2-min coast. A 35-p1 portion was careful ly taken from the top . Incubation of this sample and the 35-p1 control al iquot (not spun) at 26°C was continued unti l a total incubation time of 90 min for each sample was reached . A 25-pl portion of each was then TCA-precipitated and prepared for SDS-PAGE . With increasing preincubation time, increasing amounts of prolactin mRNA would be expected to be assembled into membrane-bound polysomes and, therefore, to be removed from the translocation mix by sedimentation . Incubation of the supernatant f luid fraction would be expected to result in the translation of al l free mRNA and mRNA engaged in free polysomes . Under the condit ions used, the amount of preprolactin made in our translation system is proportional to the amount of mRNA added to the translation . The f inal amount of preprolactin therefore is a function of the amount of translatable mRNA left in the supernatant f luid after the centri fugation step, and thereby a measure of the amount of mRNA not attached to the microsomal membrane at any given time of preincubation .