We have analyzed early phases of the cotranslational transport of the secretory protein preprolactin through the mammalian endoplasmic reticulum (ER) membrane. Following recognition of the signal sequence of the nascent polypeptide chain in the cytosol by the SRP, the chain is transferred into the membrane, where a second signal sequence recognition step takes place for which the presence in the lipid bilayer of the Sec61p complex is essential and sufficient. This step leads to a tight junction between the ribosomenascent chain complex and the Sec61p complex, and to the productive insertion of the nascent chain into the translocation site. These results show that a translocation substrate is subjected to two recognition events before being allowed to cross the ER membrane.

No abstract

Protein transport across the endoplasmic reticulum membrane can occur by two pathways, a co- and a post-translational one. In both cases, polypeptides are first targeted to translocation sites in the membrane by virtue of their signal sequences and then transported across or inserted into the phospholipid bilayer, most likely through a protein-conducting channel. Key components of the translocation apparatus have now been identified and the translocation pathways seem likely to be related to each other but mechanistically distinct. Protein transport across the bacterial inner membrane is both similar to and different from the process in eukaryotes. Other pathways of protein translocation exist that bypass the ones involving classical signal sequences.

The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.

Members of the nuclear factor (NF)-κB family of transcription factors play a crucial role in cellular activation, immune responses, and oncogenesis. In most cells, they are kept inactive in the cytosol by complex formation with members of the inhibitor of NF-κB (IκB) family, whose degradation activates NF-κB in response to diverse stimuli. In Hodgkin's lymphoma (HL), high constitutive nuclear activity of NF-κB is characteristic of the malignant Hodgkin and Reed-Sternberg (H/RS) cells, which occur at low number in a background of nonneoplastic inflammatory cells. In single H/RS cells micromanipulated from histological sections of HL, we detect clonal deleterious somatic mutations in the IκBα gene in two of three Epstein-Barr virus (EBV)-negative cases but not in two EBV-positive cases (in which a viral oncogene may account for NF-κB activation). There was no evidence for IκBα mutations in two non-HL entities or in normal germinal center B cells. This study establishes deleterious IκBα mutations as the first recurrent genetic defect found in H/RS cells, indicating a role of IκBα defects in the pathogenesis of HL and implying that IκBα is a tumor suppressor gene.

Abstract. Cotranslational translocation of proteins across the mammalian ER membrane involves, in addition to the signal recognition particle receptor and the Sec61p complex, the translocating chain-associating membrane (TRAM) protein, the function of which is still poorly understood. Using reconstituted proteoliposomes, we show here that the translocation of most, but not all, secretory proteins requires the function of TRAM. Experiments with hybrid proteins demonstrate that the structure of the signal sequence determines whether or not TRAM is needed. Features that distinguish TRAM-dependent and -independent signal sequences include the length of their charged, NH~-terminal region and the structure of their hydrophobic core. In cases where TRAM is required for translocation, it is not needed for the initial interaction of the ribosome/ nascent chain complex with the ER membrane but for a subsequent step inside the membrane in which the nascent chain is inserted into the translocation site in a protease-resistant manner. Thus, TRAM functions in a signal sequence-dependent manner at a critical, early phase of the translocation process.

Epstein-Barr virus (EBV) is IntroductionThe hallmark of the germinal center (GC) reaction is the process of somatic hypermutation (SH), which leads to B cells with a highaffinity B-cell receptor (BCR) and the abrogation of the default apoptosis pathway of GC B cells. 1 Since SH in the V genes of the rearranged immunoglobulin heavy (HC) and light chain (LC) alleles is largely random, most mutations will have a negative impact on the BCR to bind its cognate antigen. Some mutations, such as generation of a stop codon or a frame shift mutation caused by insertions or deletions, will abrogate proper translation of the immunoglobulin genes, which make up the BCR. Such mutations are lethal for the cells that harbor them: these cells are eliminated by apoptosis either because they cannot compete with cells that bind antigen more strongly or because they no longer express a BCR on their surface and therefore fail to signal. 2 It is therefore surprising that about 25% of Hodgkin/ReedSternberg (HRS) cells, which are the characteristic cells of classic Hodgkin lymphoma, carry crippling mutations in the rearranged HC or LC alleles, which preclude the expression of a functional BCR. 3 The Hodgkin tumors are clonal and derived from GC B cells because they carry originally functional light and heavy chain immunoglobulin alleles with typical modifications introduced by SH. 4,5 It is therefore likely that HRS cells with crippling mutations in the immunoglobulin genes have acquired additional transforming event(s), which enabled the cells to escape the negative selection in the GC reaction.One of the most prominent candidates in supporting this transforming event is Epstein-Barr virus (EBV), which is categorized as a class 1 carcinogen by the World Health Organization and has been found associated with B-cell lymphomas such as Hodgkin lymphoma, Burkitt lymphoma, posttransplantation lymphomas, and certain carcinomas, among others. 6 In all EBV-infected tumor cells the virus adopts a latent state, which is accompanied by the expression of a restricted number of latent viral genes. In vitro, EBV infects resting human B lymphocytes and transforms them into lymphoblastoid cell lines (LCLs); a process that is termed growth transformation and a hallmark of this virus. EBV encodes several proteins, which could play a crucial role in rescuing GC B cells from apoptosis. These B cells are inherently prone to die, and to survive must receive specific survival signals such as activation of the B cell and CD40 receptors by contact with antigen and T helper cells, respectively. These signals could be replaced by the viral proteins LMP2A and LMP1, both of which are expressed in EBV-infected HRS B cells and posttransplantation lymphomas. [7][8][9] Whereas LMP1 in many aspects resembles a constitutively active CD40 receptor, 10-12 the cytoplasmic tail of LMP2A harbors an immunoreceptor tyrosine-based activation motif (ITAM). 13 This motif has originally been identified in the coreceptors CD79A and CD79B, which make up the BCR, together with the ass...

The signal-recognition particle (SRP) is important for the targeting of many secretory and membrane proteins to the endoplasmic reticulum (ER). Targeting is regulated by three GTPases, the 54K subunit of SRP (SRP54), and the alpha- and beta-subunits of the SRP receptor. When a signal sequence emerges from the ribosome, SRP interacts with it and targets the resulting complex to the ER membrane by binding to the SRP receptor. Subsequently, SRP releases the signal sequence into the translocation channel. Here we use a complex of a ribosome with a nascent peptide chain, the SRP and its receptor, to investigate GTP binding to SRP54, and GTP hydrolysis. Our findings indicate that a ribosomal component promotes GTP binding to the SRP54 subunit of SRP. GTP-bound SRP54 is essential for high-affinity interaction between SRP and its receptor in the ER membrane. This interaction induces the release of the signal sequence from SRP, the insertion of the nascent polypeptide chain into the translocation channel, and GTP hydrolysis. The contribution of the ribosome had previously escaped detection because only synthetic signal peptides were used in the analysis.