The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling cascade known as the unfolded protein response (UPR). Although activation of the UPR is well described, there is little sense of how the response, which initiates both apoptotic and adaptive pathways, can selectively allow for adaptation. Here we describe the reconstitution of an adaptive ER stress response in a cell culture system. Monitoring the activation and maintenance of representative UPR gene expression pathways that facilitate either adaptation or apoptosis, we demonstrate that mild ER stress activates all UPR sensors. However, survival is favored during mild stress as a consequence of the intrinsic instabilities of mRNAs and proteins that promote apoptosis compared to those that facilitate protein folding and adaptation. As a consequence, the expression of apoptotic proteins is short-lived as cells adapt to stress. We provide evidence that the selective persistence of ER chaperone expression is also applicable to at least one instance of genetic ER stress. This work provides new insight into how a stress response pathway can be structured to allow cells to avert death as they adapt. It underscores the contribution of posttranscriptional and posttranslational mechanisms in influencing this outcome.

Abstract. The mitogen-activated protein (MAP) kinase signal transduction pathway represents an important mechanism by which growth factors regulate cell function. Targets of the MAP kinase pathway are located within several cellular compartments. Signal transduction therefore requires the localization of MAP kinase in each sub-cellular compartment that contains physiologically relevant substrates. Here, we show that serum treatment causes the translocation of two human MAP kinase isoforms, p40 "~k and p41 inapt, from the cytosol into the nucleus. In addition, we report that p41 m~k (but not p40 "~'k) is localized at the cell surface ruffling membrane in serum-treated cells.To investigate whether the protein kinase activity of MAP kinase is required for serum-induced redistribution within the cell, we constructed mutated kinasenegative forms of p40 ~pk and p41~. The kinasenegative MAP kinases were not observed to localize to the cell surface ruffling membrane. In contrast, the kinase-negative MAP kinases were observed to be translocated to the nucleus. Intrinsic MAP kinase activity is therefore required only for localization at the cell surface and is not required for transport into the nucleus.Together, these data demonstrate that the pattern of serum-induced redistribution of p40"~ is different from p41,,~k. Thus, in addition to common targets of signal transduction, it is possible that these MAP kinase isoforms may differentially regulate targets located in distinct sub-cellular compartments.

Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR). Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex. We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis. In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked. Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54.

High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A 2 a (hA 2 aR) receptor is active for ligandbinding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA 2 aR, as expected, and partially cleaved for hA 2 bR, hCCR5R, and hD 2L R. Ligand-binding assays conducted on the adenosine family (hA 1 R, hA 2 aR, hA 2 bR, and hA 3 R) of receptors show that hA 2 aR and hA 2 bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae.

Mitogen‐activated protein kinases (MAP kinases) are a group of closely related enzymes implicated in signal transduction pathways. We report the molecular cloning of four human proteins (p40 mapk , p41 mapk , p44 mapk and p63 mapk , with high homology to members of the MAP kinase family. Sequence analysis demonstrated that p44 mapk and p63 mapk were the products of distinct genes. However, the p40 mapk and p41 mapk were found to be related, and are likely to result from alternative processing of transcripts from a single gene. The heterogeneous expression of these human MAP kinase isoforms in different tissues may reflect the diversity of signal transduction pathways in differentiated cells.

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Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequenceindependent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex. INTRODUCTIONThe N-terminal hydrophobic signal sequence contains the sorting information that specifies transport of a protein across the rough endoplasmic reticulum (RER). Nascent polypeptides are cotranslationally translocated across the RER through an aqueous protein-conducting channel (Simon and Blobel, 1991;Crowley et al., 1994) that is comprised of the heterotrimeric Sec61 complex (Gö rlich et al., 1992;Gö rlich and Rapoport, 1993). Three to four Sec61 complexes oligomerize to form a quasi-pentagonal ring surrounding a 25Å diameter pore, that is believed to be the channel through which the nascent polypeptide traverses the membrane (Hanein et al., 1996). The Sec61 complex binds ribosomes with high affinity (K d ϭ 5 nM), hence the Sec61 complex serves a dual function acting as both the core of the translocation channel and the ribosome receptor (Gö rlich et al., 1992;Kalies et al., 1994). The molecular mechanism responsible for efficient, high fidelity targeting of the RNC to the Sec61 translocation channel has received considerable attention now that the dual function of the Sec61 complex has been appreciated. Ribosomes translating cytosolic proteins could conceivably interfere with protein translocation by nonselectively binding to the Sec61 complex. Furthermore, signal sequence-independent binding of an RNC to the Sec61 complex could result in the subsequent aberrant transport of a cytosolic protein into the lumen of the rough endoplasmic reticulum. Indeed, purified RNCs that lack signal sequences will bind to the Sec61 complex resulting in detectable, albeit inefficient, translocation of * Corresponding author: Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, 55 Lake Avenue North...