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Isbert S Wagner K Eggert S Schweitzer A Multhaup G Weggen S Kins S Pietrzik CU 《Cellular and molecular life sciences : CMLS》2012,69(8):1353-1375
The amyloid precursor protein (APP) is part of a larger gene family, which has been found to form homo- or heterotypic complexes
with its homologues, whereby the exact molecular mechanism and origin of dimer formation remains elusive. In order to assess
the cellular location of dimerization, we have generated a cell culture model system in CHO-K1 cells, stably expressing human
APP, harboring dilysine-based organelle sorting motifs [KKAA-endoplasmic reticulum (ER); KKFF-Golgi], accomplishing retention
within early secretory compartments. We show that APP exists as disulfide-bonded dimers upon ER retention after it was isolated
from cells, and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. In contrast, strong denaturing
and reducing conditions, or deletion of the E1 domain, resulted in the disappearance of those dimers. Thus we provide first
evidence that a fraction of APP can associate via intermolecular disulfide bonds, likely generated between cysteines located
in the extracellular E1 domain. We particularly visualize APP dimerization itself and identified the ER as subcellular compartment
of its origin using biochemical or split GFP approaches. Interestingly, we also found that minor amounts of SDS-resistant
APP dimers were located to the cell surface, revealing that once generated in the oxidative environment of the ER, dimers
remained stably associated during transport. In addition, we show that APP isoforms encompassing the Kunitz-type protease
inhibitor (KPI) domain exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-mediated cell aggregation of Drosophila Schneider S2-cells was isoform independent. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker
cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated
in the ER. 相似文献
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Global analysis of protein phosphorylation in yeast 总被引:1,自引:0,他引:1
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Licatalosi DD Mele A Fak JJ Ule J Kayikci M Chi SW Clark TA Schweitzer AC Blume JE Wang X Darnell JC Darnell RB 《Nature》2008,456(7221):464-469
Protein-RNA interactions have critical roles in all aspects of gene expression. However, applying biochemical methods to understand such interactions in living tissues has been challenging. Here we develop a genome-wide means of mapping protein-RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). HITS-CLIP analysis of the neuron-specific splicing factor Nova revealed extremely reproducible RNA-binding maps in multiple mouse brains. These maps provide genome-wide in vivo biochemical footprints confirming the previous prediction that the position of Nova binding determines the outcome of alternative splicing; moreover, they are sufficiently powerful to predict Nova action de novo. HITS-CLIP revealed a large number of Nova-RNA interactions in 3' untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain. HITS-CLIP, therefore, provides a robust, unbiased means to identify functional protein-RNA interactions in vivo. 相似文献