ATF6, a member of the leucine zipper protein family, can constitutively induce the promoter of glucoseregulated protein (grp) genes through activation of the endoplasmic reticulum (ER) stress element (ERSE). To understand the mechanism of grp78 induction by ATF6 in cells subjected to ER calcium depletion stress mediated by thapsigargin (Tg) treatment, we discovered that ATF6 itself undergoes Tg stress-induced changes. In nonstressed cells, ATF6, which contains a putative short transmembrane domain, is primarily associated with the perinuclear region. Upon Tg stress, the ATF6 protein level dropped initially but quickly recovered with the additional appearance of a faster-migrating form. This new form of ATF6 was recovered as soluble nuclear protein by biochemical fractionation, correlating with enhanced nuclear localization of ATF6 as revealed by immunofluorescence. Optimal ATF6 stimulation requires at least two copies of the ERSE and the integrity of the tripartite structure of the ERSE. Of primary importance is a functional NF-Y complex and a high-affinity NF-Y binding site that confers selectivity among different ERSEs for ATF6 inducibility. In addition, we showed that YY1 interacts with ATF6 and in Tg-treated cells can enhance ATF6 activity. The ERSE stimulatory activity of ATF6 exhibits properties distinct from those of human Ire1p, an upstream regulator of the mammalian unfolded protein response. The requirement for a high-affinity NF-Y site for ATF6 but not human Ire1p activity suggests that they stimulate the ERSE through diverse pathways.

Yeast Hac1 (yHac1), the transcription factor that binds and activates the unfolded protein response element of endoplasmic reticulum (ER)-chaperone gene promoters, only accumulates in stressed cells after an unconventional splicesosome-free mRNA processing step and escape from translation block. In determining whether the novel regulatory mechanisms for yHac1 are conserved in mammalian cells, we discovered a unique unfolded protein response element-like sequence within the endoplasmic reticulum stress element 163, one of the three ER stress elements recently identified in the rat grp78 promoter. The unspliced form of yHac1 is stably expressed in nonstressed mammalian cells and is as active as the spliced form in stimulating the promoter activities of grp genes. Further, the yHac1 mRNA is not processed in response to ER stress in mammalian cells. We identified a CCAGC motif as the yHac1 binding site, which is contained within a YY1 binding site previously shown to be important for mammalian UPR. Dissection of the yHac1 and the YY1 binding sites uncovered specific contact points for an activator protein predicted to be the mammalian homolog of yHac1, the activity of which can be stimulated by YY1. A model of the conserved and unique features of the yeast and mammalian unfolded protein response transcription machinery is proposed. The endoplasmic reticulum (ER)1 is an essential organelle for cell viability. In addition to serving as a major intracellular store for calcium and the production site for lipids and sterols, ER is the cellular organelle for the synthesis, assembly, and glycosylation of proteins that are destined for secretion or transport to the cell surface. Perturbations of ER result in stress signaling from the ER to the nucleus, leading to the activation of specific sets of genes (1, 2). Conserved from yeast to human is a mechanism referred to as the unfolded protein response (UPR) (3, 4). In Saccharomyces cerevisiae, several key components of the UPR have been identified through genetic analysis and biochemical characterizations. Thus, the yeast UPR is mediated by a novel signaling pathway that requires ER stress-inducible splicing of the primary transcript of Hac1, also referred to as Ern4p (5, 6). The open reading frame of the Hac1 gene encodes a protein of 230 amino acids (aa), whereas the active transcription factor is a 238-aa protein. In nonstressed yeast cells, the unspliced Hac1 mRNA is prevented from translation into the 230-aa protein by the presence of a translation attenuator in a 252-bp intron (6, 7). Upon induction of the UPR, unconventional splicing mediated by Ire1p results in the elimination of the intronic sequence, which contains the codons encoding the last 10 aa of the 230-aa Hac1 and part of the 3Ј-untranslated region. Although the last 10 aa of 230-aa Hac1 is deleted through this process, the newly spliced Hac1 mRNA contains an additional exon of 18 aa, resulting in the translation of the 238-aa Hac1. Finally, in yeast cells, 230-aa Hac1 and 238-aa Hac1 produced from exogenous...

Amylase activity variation in the guts of several model organisms appears to be explained by amylase gene copy number variation. We tested the hypothesis that amylase gene copy number is always elevated in animals with high amylolytic activity. We therefore sequenced the amylase genes and examined amylase gene copy number in prickleback fishes (family Stichaeidae) with different diets including two species of convergently evolved herbivores with the elevated amylase activity phenotype. We found elevated amylase gene copy number (six haploid copies) with sequence variation among copies in one herbivore (Cebidichthys violaceus) and modest gene copy number (two to three haploid copies) with little sequence variation in the remaining taxa, which included herbivores, omnivores, and a carnivore. Few functional differences in amylase biochemistry were observed, and previous investigations showed similar digestibility among the convergently evolved herbivores with differing amylase genetics. Hence, the phenotype of elevated amylase activity can be achieved by different mechanisms (i.e., elevated expression of fewer genes, increased gene copy number, or expression of more efficient amylase proteins) with similar results. Phylogenetic and comparative genomic analyses of available fish amylase genes show mostly lineage-specific duplication events leading to gene copy number variation, although a whole-genome duplication event or chromosomal translocation may have produced multiple amylase copies in the Ostariophysi, again showing multiple routes to the same result.

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