Joseph F. Sambrook

A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus

In eukaryotic cells, the accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers a signaling pathway from the ER to the nucleus. Several yeast mutants defective in this pathway map to the ERN1 gene, which protects cells from lethal consequences of stress by signaling for increased expression of BiP and other ER proteins. ERN1 encodes a 1115 amino acid transmembrane protein (Ern1p) whose glycosylated N-terminal portion is located inside microsomes and whose cytoplasmic C-terminal portion carries an essential protein kinase activity. We postulate that Ern1p is the proximal sensor of events in the ER and that binding of ligand causes transduction of information across the ER membrane, leading to activation of a specific set of transcription factors.

Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.

S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP

The endoplasmic reticulum (ER) of mammalian cells contains a 78 kd protein (BiP) that is believed to assist in the folding of secretory and transmembrane proteins. We have used a cDNA encoding mouse BiP to isolate the homologous gene from S. cerevisiae, which encodes a sequence of 682 amino acids, 431 of which are identical to mouse BiP. Like its mammalian counterpart, yeast BiP is encoded by an HSP70-like gene whose transcription is stimulated by the presence of unfolded polypeptides in the ER. The gene encoding yeast BiP is essential for cell growth and, unexpectedly, is identical to the recently cloned KAR2 gene. Expression of mammalian BiP in S. cerevisiae can complement a mutant allele of KAR2 that is temperature sensitive for growth and nonconditionally defective for karyogamy. These results suggest that deficiencies in BiP may cause generalized failure of protein folding in the ER, leading to pleiotropic effects on cellular metabolism.

A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins.

The KAR2 gene of Saccharomyces cerevisiae codes for an essential chaperone protein (BiP) that is localized in the lumen of the endoplasmic reticulum (ER). The high basal rate of transcription of KAR2 is increased transiently by heat shock: prolonged induction occurs when unfolded proteins accumulate in the ER. Three cis‐acting elements in the KAR2 promoter control expression of KAR2: (i) a GC‐rich region that contributes to the high level of constitutive expression, (ii) a functional heat shock element (HSE) and (iii) an element (UPR) that is involved in the induction of BiP mRNA by unfolded proteins. By analyzing internal deletion mutants of the KAR2 promoter, we demonstrate here that these three elements regulate transcription of KAR2 independently. Furthermore, the 22 bp UPR element causes a heterologous (CYC1) promoter to respond to the presence of unfolded proteins in the ER. Extracts of both stressed and unstressed yeast cells contain proteins that bind specifically to synthetic HSE and UPR elements and retard their migration through gels. Binding proteins specific for the UPR element can be fractionated by ammonium sulfate precipitation. Two of the proteins UPRF‐1 and UPRF‐2 (which is apparently a proteolytic degradation product of UPRF‐1) bind inefficiently to mutant versions of the UPR that are unable to confer responsiveness to unfolded proteins to the (CYC1) promoter. UPRF‐1 therefore displays the properties expected of a transcription factor that is involved in the sustained response of the KAR2 promoter to unfolded proteins in the ER. These experiments show that yeast cells can activate a transcription factor that stimulates expression of a nuclear gene in response to the accumulation of unfolded proteins in another cellular compartment.

The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum.

The endoplasmic reticulum (ER) of eukaryotic cells contains an abundant 78,000-Da protein (BiP) that is involved in the translocation, folding, and assembly of secretory and transmembrane proteins. In the yeast Saccharomyces cerevisiae, as in mammalian cells, BiP mRNA is synthesized at a high basal rate and is further induced by the presence of increased amounts of unfolded proteins in the ER. However, unlike mammalian BiP, yeast BiP is also induced severalfold by heat shock, albeit in a transient fashion. To identify the regulatory sequences that respond to these stimuli in the yeast KAR2 gene that encodes BiP, we have cloned a 1.3-kb segment of DNA from the region upstream of the sequences coding for BiP and fused it to a reporter gene, the Escherichia coli beta-galactosidase gene. Analysis of a series of progressive 5 truncations as well as internal deletions of the upstream sequence showed that the information required for accurate transcriptional regulation of the KAR2 gene in S. cerevisiae is contained within a approximately 230-bp XhoI-DraI fragment (nucleotides -245 to -9) and that this fragment contains at least two cis-acting elements, one (heat shock element [HSE]) responding to heat shock and the other (unfolded protein response element [UPR]) responding to the presence of unfolded proteins in the ER. The HSE and UPR elements are functionally independent of each other but work additively for maximum induction of the yeast KAR2 gene. Lying between these two elements is a GC-rich region that is similar in sequence to the consensus element for binding of the mammalian transcription factor Sp1 and that is involved in the basal expression of the KAR2 gene. Finally, we provide evidence suggesting that yeast cells monitor the concentration of free BiP in the ER and adjust the level of transcription of the KAR2 gene accordingly; this effect is mediated via the UPR element in the KAR2 promoter.

The involvement of calcium in transport of secretory proteins from the endoplasmic reticulum.

Article synthese sur le mecanisme permettant le transport des proteines secretees a partir du reticulum endoplasmique; interet porte au calcium et a son role dans ce transport biologique

The expression of influenza virus hemagglutinin in the pancreatic β cells of transgenic mice results in autoimmune diabetes

Insulin-dependent diabetes mellitus results from the autoimmune destruction of the insulin-producing beta cells of the pancreatic islets. The target antigen(s) involved in this immunopathological process has not been identified. Our strategy was to determine whether expression of a novel surface antigen by murine pancreatic beta cells would result in insulin-dependent diabetes mellitus. We have generated lines of transgenic mice (RIP-HA) that express the hemagglutinin of the A/Japan/305/57 strain of influenza virus on their insulin-producing beta cells. Hyperglycemia developed in mice derived from all three founders at a frequency varying from 13% to 27%, and was associated with lymphocytic infiltration of the islets and a humoral response against beta cell antigens, including hemagglutinin. These results suggest that the RIP-HA mice should provide a useful system in which to study the cellular interactions involved in the induction of self-tolerance and autoimmunity.

The cellular response to unfolded proteins: intercompartmental signaling

Both prokaryotic and eukaryotic cells respond to the accumulation of unfolded proteins by increasing the transcription of genes encoding molecular chaperones and other stress-responsive proteins. Different sets of genes are activated when particular cellular compartments are burdened with unfolded proteins. Cells thus maintain mechanisms to monitor changes in the concentration of unfolded proteins not only in the cytosol, but also in membrane-bound extracytoplasmic compartments. During the past year, work in yeast has identified a transmembrane receptor that appears to play a pivotal role in the regulation of protein folding. This receptor monitors the concentration of available chaperone molecules in the endoplasmic reticulum and transmits a signal to the cytosol to activate the transcription of nuclear genes encoding chaperones that are localized in the endoplasmic reticulum. Work using Escherichia coli suggests that prokaryotes also contain an intercompartmental unfolded protein signaling pathway, in this case from the periplasmic space or outer membrane to the cytoplasm.

Variants of human tissue-type plasminogen activator that lack specific structural domains of the heavy chain.

The heavy chain of tissue plasminogen activator (t‐PA) consists of four domains [finger, epidermal‐growth‐factor (EGF)‐like, kringle 1 and kringle 2] that are homologous to similar domains present in other proteins. To assess the contribution of each of the domains to the biological properties of the enzyme, site‐directed mutagenesis was used to generate a set of mutants lacking sequences corresponding to the axons encoding the individual structural domains. The mutant proteins were assayed for their ability to hydrolyze artificial and natural substrates in the presence and absence of fibrin, to bind to lysine‐Sepharose and to be inhibited by plasminogen activator inhibitor‐1. All the deletion mutants exhibit levels of basal enzymatic activity very similar to that of wild‐type t‐PA assayed in the absence of fibrin. A mutant protein lacking the finger domain has a 2‐fold higher affinity for plasminogen than wild‐type t‐PA, while the mutant that lacks both finger and EGF‐like domains is less active at low concentrations of plasminogen. Mutants lacking both kringles neither bind to lysine‐Sepharose nor are stimulated by fibrin. However, mutants containing only one kringle (either kringle 1 or kringle 2) behave indistinguishably from one another and from the wild‐type protein. We conclude that kringle 1 and kringle 2 are equivalent in their ability to mediate stimulation of catalytic activity by fibrin.

Expression and regulation of endothelin precursor mRNA m avascular human amnion

Posttranslational processing of preproendothelin in endothelial cells gives rise to endothelin, a 21 amino acid polypeptide that is a potent vasoconstrictor. Endothelin production is believed to be mediated principally by transcriptional mechanisms. Previously, preproendothelin mRNA expression has been detected only in vascular endothelial tissue and cells. In this study, we found that preproendothelin mRNA is expressed in an avascular human tissue, namely, amnion, an extraembryonic fetal membrane. Preproendothelin mRNA was not detected in avascular chorion laeve tissue (also an extraembryonic fetal membrane), in the highly vascularized fetal trophoblast, or in maternal uterine tissues. Furthermore, we found that preproendothelin gene expression is retained in human amnion cells maintained in primary monolayer culture. Using the amnion cells in primary monolayer culture to investigate the regulation of preproendothelin mRNA expression, we found that epidermal growth factor (EGF) and interleukin-1 (IL-1) act to stimulate preproendothelin mRNA levels; in addition, the induction of preproendothelin mRNA by either of these agents is enhanced upon simultaneous treatment with cycloheximide. These findings are indicative that preproendothelin gene expression in amnion is regulated positively by EGF and IL-1 and that inhibition of protein synthesis leads to superinduction of preproendothelin mRNA. In human umbilical cord endothelial cells, neither IL-1 nor EGF stimulate preproendothelin mRNA expression but inhibition of protein synthesis does lead to increased levels of preproendothelin mRNA. The amnion, therefore, provides a useful system for expansion of our understanding of the tissue specific expression and regulation of preproendothelin mRNA.