Translocation in yeast and mammalian cells: not all signal sequences are functionally equivalent

In Saccharomyces cerevisiae, nascent carboxypeptidase Y (CPY) is directed into the endoplasmic reticulum by an NH2-terminal signal peptide that is removed before the glycosylated protein is transported to the vacuole. In this paper, we show that this signal peptide does not function in mammalian cells: CPY expressed in COS-1 cells is not glycosylated, does not associate with membranes, and retains its signal peptide. In a mammalian cell-free protein-synthesizing system, CPY is not translocated into microsomes. However, if the CPY signal is either mutated to increase its hydrophobicity or replaced with that of influenza virus hemagglutinin, the resulting precursors are efficiently translocated both in vivo and in vitro. The implications of these results for models of signal sequence function are discussed.     

Getting a grip on adhesion: Cadherin switching and collagen signaling

Epithelial-mesenchymal transition (EMT) is a developmental biological process that is hijacked during tumor progression. Cadherin switching, which disrupts adherens junctions and alters cadherin-associated signaling pathways, is common during EMT. In many tumors, substantial extracellular matrix (ECM) is deposited. Collagen is the most abundant ECM constituent and it mediates specific signaling pathways by binding to integrins and discoidin domain receptors (DDRs). The interaction of the collagen receptors results in activation of signaling pathways that promote tumor progression including an induction of the cadherin switching. DDR inhibitors have demonstrated anticancer therapeutic efficacy preclinically by inhibiting the collagen signaling. Understanding how collagen signaling impacts cellular processes including EMT and cadherin switching is of great interest especially given the strong interest in stromal targeted therapies for desmoplastic cancers.     

Terminal beta-D-galactofuranosyl epitopes recognized by antibodies that inhibit Trypanosoma cruzi internalization into mammalian cells

Polyclonal antisera to 80 - 90-kDa and to 50 - 60-kDa polypeptides of tissue-culture trypomastigotes which inhibit the interiorization of trypomastigotes of the Y strain (up to 70%) and of metacyclic trypomastigotes of the CL-14 clone (up to 50%) in cultured mammalian cells, were obtained. Both sera immunoprecipitate surface polypeptides of 90 kDa, 80 kDa, 72 kDa and 58 kDa in trypomastigotes and of 80 kDa, 77 kDa and 74 kDa in metacyclic trypomastigotes. These antigens are glycoproteins with affinity for concanavalin A. The antibodies (IgG class) of the inhibitory sera are mainly directed against carbohydrate epitopes, which were identified as being beta-D-galactofuranosyl units by radioimmunoinhibition assays. The direct involvement of the beta-D-galactofuranosyl unit in the process of parasite infection was verified using the synthetic disaccharide beta-D-galactofuranose (1----3)-alpha-D-mannopyranose which promoted, at 1 mg/ml concentration, 50% inhibition of internalization.     

Hippo signaling in mammalian stem cells

Over the past decade, the Hippo signaling cascade has been linked to organ size regulation in mammals. Indeed, modulation of the Hippo pathway can have potent effects on cellular proliferation and/or apoptosis and a deregulation of the pathway often leads to tumor development. Importantly, emerging evidence indicates that the Hippo pathway can modulate its effects on tissue size by the regulation of stem and progenitor cell activity. This role has recently been associated with the central position of the pathway in sensing spatiotemporal or mechanical cues, and translating them into specific cellular outputs. These results provide an attractive model for how the Hippo cascade might sense and transduce cellular ‘neighborhood’ cues into activation of tissue-specific stem or progenitors cells. A further understanding of this process could allow the development of new therapies for various degenerative diseases and cancers. Here, we review current and emerging data linking Hippo signaling to progenitor cell function.     

Mitochondrial death pathways in yeast and mammalian cells

In mammals, mitochondria are important mediators of programmed cell death, and this process is often regulated by Bcl-2 family proteins. However, a role for mitochondria-mediated cell death in non-mammalian species is more controversial. New evidence from a variety of sources suggests that mammalian mitochondrial fission/division proteins also have the capacity to promote programmed cell death, which may involve interactions with Bcl-2 family proteins. Homologues of these fission factors and several additional mammalian cell death regulators are conserved in flies, worms and yeast, and have been suggested to regulate programmed cell death in these species as well. However, the molecular mechanisms by which these phylogenetically conserved proteins contribute to cell death are not known for any species. Some have taken the conserved pro-death activity of mitochondrial fission factors to mean that mitochondrial fission per se, or failed attempts to undergo fission, are directly involved in cell death. Other evidence suggests that the fission function and the cell death function of these factors are separable. Here we consider the evidence for these arguments and their implications regarding the origins of programmed cell death.     

Size control in growing yeast and mammalian cells

Background  

In a recent publication it was claimed that cultured mammalian cells, in contrast to yeasts, maintain a constant size distribution in the population without a size checkpoint. This inference may be challengeable.     

Differential ER exit in yeast and mammalian cells

The coat complex COPII forms vesicles at the endoplasmic reticulum to transport a variety of cargo proteins to the Golgi structure. Recent biochemical and structural studies reveal the molecular mechanism of cargo protein recognition by COPII components. Furthermore, there are at least two distinct ER-to-Golgi transport carrier structures carrying different cargo proteins in yeast and mammalian cells, suggesting several distinct mechanisms for the concentration, selection and exit of cargo proteins from the ER. It will be essential to follow the dynamics of transitional ER sites and cargo protein concentration within the ER in order to understand how these transport processes occur in living cells.     

Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells.

In many viruses and transposons, expression of some genes requires alternative reading of the genetic code, also called recoding. Such events depend on specific mRNA sequences and can lead to read through of an in-frame stop codon or to +1 or -1 frameshifting. Here, we addressed the issue of conservation of recoding rules between the yeast Saccharomyces cerevisiae and mammalian cells by establishing a versatile vector that can be used to study recoding in both species. We first assessed this vector by analysing the site of +1 frameshift of the Ty1 transposon. Two sequences from higher organisms were then tested in both yeast and mammalian cells: the gag-pol junction of human immunodeficiency virus type 1 (HIV-1) (a site of -1 frameshift), and the stop codon region of the replicase cistron from the tobacco mosaic virus (a site of UAG read through). We show that both sequences direct a high level of recoding in yeast. Furthermore, different mutations of the target sequences have similar effects on recoding in yeast and in mouse cells. Most notably, a strong decrease of frameshifting was observed in the absence of the HIV-1 stem-loop stimulatory signal. Taken together, these data suggest that mechanisms of some recoding events are conserved between lower and higher eukaryotes, thus allowing the use of S. cerevisiae as a model system to study recoding on target sequences from higher organisms.     

5''-flanking sequences that inhibit in vitro transcription of a xenopus laevis tRNA gene

We determined the nucleotide sequences of all coding regions and a significant part of the flanking regions of the chicken c-src gene, which is a cellular homolog of the v-src gene of Rous sarcoma virus. The c-src gene consists of 12 exons; the boundaries of the exons were determined by assuming that the amino acid sequence of its product, pp60c-src, is basically the same as that of pp60v-src. The deduced amino acid sequence of pp60c-src was very similar to that of pp60v-src, but the last 19 carboxy-terminal amino acids of pp60c-src were replaced by a new set of 12 amino acids of pp60v-src. The sequence encoding the carboxy-terminal sequence of pp60v-src was found 900 bp downstream from the termination codon of the c-src gene. We suggest that the c-src sequence was captured by a virus through recombination at both sides of the c-src gene, and that the recombinations occurred at the level of proviral DNA.     

Endoplasmic reticulum stress-sensing mechanisms in yeast and mammalian cells

Upon endoplasmic reticulum (ER) stress, ER-located transmembrane stress sensors evoke diverse protective responses. Although ER stress-dependent activation of the sensor proteins is partly explained through their negative regulation by the ER-located chaperone BiP under non-stress conditions, each of the sensors is also regulated by distinct mechanism(s). For instance, yeast Ire1 is fully activated via its direct interaction with unfolded proteins accumulated in the ER. This insight is consistent with a classical notion that unfolded proteins per se trigger ER-stress responses, while various stress stimuli also seem to activate individual sensors independently of unfolded proteins and in a stimuli-specific manner. These properties may account for the different responses observed under different conditions in mammalian cells, which carry multiple ER-stress sensors.     

Oligodendrocyte precursor cells: Reactive cells that inhibit axon growth and regeneration

Oligodendrocyte precursor cells (OPCs) are a newly recognized glial component of the adult central nervous system of unknown function. Antibodies against the NG2 chondroitin sulfate proteoglycan have been useful tools to identify these cells in intact tissue. Here we review studies that show that OPCs react to several types of experimentally induced brain injury. Injury stimulates OPCs to re-enter the cell cycle, divide, and accumulate at the site of damage. OPCs, together with microglia and astrocytes, form the glial scar. Glial scars are thought to inhibit or prevent axonal regeneration and reactive OPCs contribute to this inhibition by producing growth-inhibiting chondroitin sulfate proteoglycans, particularly NG2. In developing animals, NG2 is found in areas, such as the perinotochordal mesenchyme, that are avoided by growing motor and sensory axons. Within the developing CNS, NG2-expressing cells surround the developing optic chiasm and tract and separate it from the overlying diencephalon. Thus, NG2-expressing cells are well positioned to inhibit axonal growth from developing as well as regenerating neurons.     

Genetic dissection of the signaling domain of a mammalian steroid receptor in yeast.

The mechanism of signal transduction by steroid receptor proteins is complex and not yet understood. We describe here a facile genetic strategy for dissection of the rat glucocorticoid receptor "signaling domain," a region of the protein that binds and transduces the hormonal signal. We found that the characteristics of signal transduction by the receptor expressed in yeast were similar to those of endogenous receptors in mammalian cells. Interestingly, the rank order of particular ligands differed between species with respect to receptor binding and biological efficacy. This suggests that factors in addition to the receptor alone must determine or influence ligand efficacy in vivo. To obtain a collection of receptors with distinct defects in signal transduction, we screened in yeast an extensive series of random point mutations introduced in that region in vitro. Three phenotypic classes were obtained: one group failed to bind hormone, a second displayed altered ligand specificity, and a third bound hormone but lacked regulatory activity. Our results demonstrate that analysis of glucocorticoid receptor action in yeast provides a general approach for analyzing the mechanism of signaling by the nuclear receptor family and may facilitate identification of non-receptor factors that participate in this process.     

Three yeast proteins that specifically inhibit yeast proteases A, B, and C.

Baker's yeast was found to contain inhibitors of yeast proteases A and C. These two proteins were partially purified, characterized, and compared with the previously described inhibitor of protease B. The A and B inhibitors were very thermostable and were extracted from intact yeast cells at 9k C. The A inhibitor appeared to be a protein with a molecular weight of about 22,000 which could be dissociated into two monomers or chains, both of which had a molecular weight of approximately 11,000. The protease C (carboxypeptidase Y)-inhibitor complex was purified and then partially disociated on an ion-exchange column. The free protease C inhibitor was very unstable, possibly because of destruction by a contaminating protease. Each inhibitor was specific for its corresponding protease and each inhibition was competitive. Whereas proteases A, B, and C destroyed the B inhibitor, only protease B had a pronounced destructive effect on the protease A inhibitor. Pepstatin was found to be a selective inhibitor of protease A, whereas chymostatin and antipain specifically inhibited protease B.     

Tetrahymena micronuclear sequences that function as telomeres in yeast.

We explored the ability of S. cerevisiae to utilize heterologous DNA sequences as telomeres by cloning germline (micronuclear) DNA from Tetrahymena thermophila on a linear yeast plasmid that selects for telomere function. The only Tetrahymena sequences that functioned in this assay were (C4A2)n repeats. Moreover, these repeats did not have to be derived from Tetrahymena telomeres, although we show that micronuclear telomeres (like macronuclear telomeres) of Tetrahymena terminate in (C4A2)n repeats. Chromosome-internal restriction fragments carrying (C4A2)n repeats also stabilized linear plasmids and were elongated by yeast telomeric repeats. In one case, the C4A2 repeat tract was approximately 1.5 kb from the end of the genomic Tetrahymena DNA fragment that was cloned, but this 1.5 kb of DNA was missing from the linear plasmid. Thus, yeast can utilize internally located tracts of telomere-like sequences, after the distal DNA is removed. The data provide an example of broken chromo-some healing, and underscore the importance of the telomeric repeat structure for recognition of functional telomeric DNA in vivo.