Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane

Abstract

| We have reproduced the process of protein transport across and of protein integration into the mammalian endoplasmic reticulum membrane by the use of proteoliposomes reconstituted from pure phospholipids and purified membrane proteins. The transport of some proteins requires only two membrane protein complexes: the signal recognition particle receptor, needed for targeting of a nascent chain to the membrane, and a novel complex, the Sec61p complex, that consists of Sec61p and two smaller polypeptides. The translocation of other proteins also needs the presence of the translocating chain-associating membrane (TRAM) protein. The integration of two membrane proteins of different topologies into the membrane does not require additional components. These results indicate a surprising simplicity of the basic translocation machinery. They suggest that the Sec61p complex binds the ribosome during translocation and forms the postulated protein-conducting channel.

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

“…In eukaryotes, mRNA encoding secreted and membrane proteins can be targeted to the ER co-translationally by the signal recognition particle 1,2 and can be maintained on the ER via the direct interactions between ribosomes and translocons during the translation 3,4 . However, whether mRNAs can be targeted and maintained on the ER independent of either ribosomes or translation was unclear until very recently.…”

Les gènes et les protéines qui contrôlent la voie de sécrétion

“…En contraste avec la règle classique de translocation co-traductionnelle découverte par Blobel, Peter Walter, un protégé de Blobel, découvrit qu'au moins un substrat, le précurseur du facteur α, la phéromone de conjugaison de la levure, pouvait traverser la membrane du RE après l'achèvement de la traduction (Gorlich & Rapoport, 1993). On supposait que quelque chose d'extrinsèque ou d'intrinsèqueà ce précurseur le maintenait sous une forme qui pouvait facilement se déplier pendant la translocation.…”

The major pathways of protein translocation across membranes

“…Sbh1 and Sec61b are also homologous. The Sec61 complexes both in mammals and yeast serve as ribosome receptors as well (Görlich & Rapoport 1993;Panzner et al 1995). Other integral membrane components like SecD/ SecF in E. coli, Sec62/Sec63/Sec71/Sec72 (Sec63 complex) in yeast, and TRAM in mammals do not function by themselves, but they either stimulate, regulate, or confer specificity upon translocation.…”

“…The GTPase activities of SRP54 and the SRP receptor subunits somehow allow the recycling of SRP (Walter & Johnson 1994). It is most likely that Sec61 , a central translocator component in the ER membrane, serves as the ribosome receptor (Görlich & Rapoport 1993) and allows the formation of a continuous aqueous environment from the 'translocation channel' to the peptidyltransferase centre (Crowley et al 1994).…”

“…This experimental system, in combination with positionspecific modification of nascent chain amino acids by crosslinking or fluorescence probes, allows detailed examination of the nascent chain environment, such as proteins in the proximity of the translocating polypeptide (Mothes et al 1994) or the hydropathic nature of the environment that the residues encounter during translocation (Crowley et al 1993). Finally, 'real biochemistry', such as purification of the machinery components and their reconstitution into proteoliposomes of defined composition, has now been achieved both in the E. coli and the ER systems Douville et al 1995;Görlich & Rapoport 1993;Panzner et al 1995). These studies have revealed a number of necessary players and their modes of actions in considerable detail.…”