Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.26 (invertase)
 4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The membrane topology of two alkane-inducible cytochromes P450 from the yeast Candida tropicalis, alk1 and alk2, was tested by construction of fusion proteins with part of invertase and histidinol dehydrogenase (invHIS4C) and expression in a Saccharomyces cerevisiae his4 mutant. Depending on the localization of invHIS4C on the endoplasmic reticulum (ER) cytoplasmic or luminal side, the enzyme converts histidinol to histidine and allows the his4 yeast strain to grow on histidinol-supplemented medium. The N-terminal segments of alk1 and alk2 were fused to invHIS4C at three different locations that follow the first alk1 and alk2 transmembrane domains or a second putative transmembrane domain of alk1. The combination of this in vivo assay with subcellular immunoprecipitations of the expressed fusion proteins allowed us to establish that both P450s contain only one transmembrane domain with their N-terminus located in the ER lumen. Deletions performed in these fusion proteins removing the first transmembrane domain of alk1 (delta TM) resulted in a less efficient targeting to the ER membrane but did not prevent their insertion in these membranes. Furthermore deletion of a negatively charged peptide preceding the first alk1 transmembrane domain (delta L) in an invHIS4C protein fused after this domain caused the N-terminal to have a positive net charge and to be oriented in the cytoplasm thus translocating the remaining protein into the ER lumen. The presence of the second hydrophobic segment, however, prevented the complete translocation of this fusion protein into the ER lumen. This study describes the first assessment of P450 membrane topology using an in vivo technique.
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PMID:Probing the membrane topology of Candida tropicalis cytochrome P450. 837 86

Overproduction of an endoplasmic reticulum (ER)-resident membrane protein (cytochrome P450 52A3) and of a secretory protein (invertase) was used to study the regulation of the luminal ER protein Kar2p under conditions that lead to ER proliferation and secretory overload, respectively. In both cases we found (i) a significant increase of Kar2 protein and mRNA levels, (ii) a transcriptional regulation based on the the function of the 22 bp unfolded-protein-response element of the KAR2 promoter and (iii) an essential role of the transmembrane kinase Ire1p for upregulation of KAR2 gene expression. These results show that the same mechanism operates when KAR2 induction is triggered by overproduction of cytochrome P450 or invertase and that this mechanism shares the known features of the unfolded-protein-response pathway. Disruption of the IRE1 gene resulted in a marked decrease of the invertase protein levels produced. In contrast, a functional IRE1 gene was not required to reach high-level production of the integral membrane protein cytochrome P450 52A3, Moreover, IRE1 gene disruption did not prevent P450-induced ER proliferation. We suggest that Ire1p-mediated KAR2 induction is, in the case of cytochrome P450 52A3 overproduction, a process which follows on ER proliferation, thereby monitoring the increase of ER size and adjusting the level of Kar2p accordingly.
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PMID:Inducible membranes in yeast: relation to the unfolded-protein-response pathway. 936 46