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AC-terminal KDEL-like motif prevents secretion of soluble endoplasmic reticulum (ER)–resident proteins. This motif interacts with KDEL receptors localized in the intermediate compartment and Golgi apparatus. Such binding triggers retrieval back to the ER via a coat protein I–dependent pathway. To date, two human KDEL receptors have been reported. Here, we report the Golgi localization of a third human KDEL receptor. Using a reporter construct system from a screen of 152 variants, we identified 35 KDEL-like variants that result in efficient ER localization but do not match the current Prosite motif for ER localization ([KRHQSA]-[DENQ]-E-L). We cloned 16 human proteins with one of these motifs and all were found in the ER. A subsequent screen by bimolecular fluorescence complementation determined the specificities of the three human KDEL receptors. Each KDEL receptor has a unique pattern of motifs with which it interacts. This suggests a specificity in the retrieval of human proteins that contain different KDEL variants.

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Hypoxia and loss of cell polarity are common features of malignant carcinomas. Hypoxia-inducible factor 1 (HIF1) is the major regulator of cellular hypoxia response and mediates the activation of ∼300 genes. Increased HIF1 signaling is known to be associated with epithelial-mesenchymal transformation. Here, we report that hypoxia disrupts polarized epithelial morphogenesis of MDCK cells in a HIF1α-dependent manner by modulating the transforming growth factor-β (TGFβ) signaling pathway. Analysis of potential HIF1 targets in the TGFβ pathway identified the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transmembrane glycoprotein related to the type I receptors of the TGFβ family, whose expression was essentially lost in HIF1-depleted cells. Similar to what was observed in HIF1-deficient cells, BAMBI-depleted cells failed to efficiently activate TGFβ signaling and retained epithelial polarity during hypoxia. Taken together, we show that hypoxic conditions promote TGFβ signaling in a HIF1-dependent manner and BAMBI is identified in this pathway as a novel HIF1-regulated gene that contributes to hypoxia-induced loss of epithelial polarity.

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Ciliated protists are model organisms for a number of molecular phenomena including telomerase function, self-splicing introns, and an RNA interference-related mechanism in programmed DNA elimination. Despite this relevance, our knowledge about promoters and transcriptional regulation in these organisms is very limited. The macronuclear genome of stichotrichous ciliates consists of minichromosomes which typically encode a single gene. The 5 nontranscribed spacers are usually no longer than 400 bp and highly suitable for promoter characterizations. We used microinjection of two artificial and differently tagged ␣1 tubulin minichromosomes into the macronucleus of Stylonychia lemnae as a means to characterize in detail the corresponding promoter. Clonal cell lines that stably maintained both minichromosomes were generated, enabling comparative expression analysis by primer extension assays. Deletion and block substitution mutations of one of the minichromosomes revealed a TATA-like element, a putative initiator element, and two distinct upstream sequence elements (USEs). Determination of transcription initiation sites and a sequence alignment indicated that both TATA-like and initiator elements are conserved components of S. lemnae minichromosomes, whereas the USEs appear to be specific for the ␣1 tubulin minichromosome. The ␣2 tubulin minichromosome promoter is very short, comprising the two proximal elements but not the USEs. Despite the latter finding, up-regulation of ␣-tubulin expression in cells treated with concanavalin A activated the ␣2 but not the ␣1 tubulin promoter. These results therefore show that gene expression regulation in S. lemnae occurs at the level of transcription initiation on the basis of structurally different promoters.In the past, research on ciliates has revealed generally important discoveries such as self-splicing introns (18), telomerase (9), and, recently, small RNAs involved in DNA elimination (26). Despite this relevance and despite evidence that ciliates regulate their genes at the transcriptional level (2, 36, 37), determinants of transcription in these organisms have not been a major focus. In Tetrahymena thermophila, promoter elements have been experimentally identified in the rRNA gene (6, 25), the telomerase RNA gene (11), and the RAD51 gene (35). However, no detailed analysis of a ciliate promoter has been achieved thus far. In ciliates, gene expression is restricted to macronuclei, which develop from micronuclei after conjugation. In this process, micronuclear DNA is fragmented, in part eliminated, and macronucleus-destined DNA is amplified (reviewed in reference 15). In stichotrichous ciliates, the extent of these DNA manipulations is extreme. For example, in Stylonychia lemnae, more than 90% of the micronuclear DNA is eliminated, DNA fragmentation leads to gene-sized minichromosomes, and DNA amplification results in gene-specific copy numbers that can exceed 100,000 per cell (12,38). A typical macronuclear minichromosome in these organisms harbors a single gene and very ...

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