Faculty Opinions recommendation of Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays.

2002 ◽  
Author(s):  
John Patrick
Keyword(s):  
Plasma Membrane ◽  
Intermolecular Interactions ◽  
Functional Assays ◽  
Sucrose Transporters ◽  
Ubiquitin System ◽  
Split Ubiquitin

scholarly journals Intra- and Intermolecular Interactions in Sucrose Transporters at the Plasma Membrane Detected by the Split-Ubiquitin System and Functional Assays

Structure ◽  
2002 ◽  
Vol 10 (6) ◽  
pp. 763-772 ◽  
Author(s):  
Anke Reinders ◽  
Waltraud Schulze ◽  
Safia Thaminy ◽  
Igor Stagljar ◽  
Wolf B. Frommer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intermolecular Interactions ◽  
Functional Assays ◽  
Sucrose Transporters ◽  
Ubiquitin System ◽  
Split Ubiquitin

Interaction of the endoplasmic reticulum α1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system

2001 ◽  
Vol 114 (24) ◽  
pp. 4629-4635
Author(s):  
Michel J. Massaad ◽  
Annette Herscovics
Keyword(s):  
Endoplasmic Reticulum ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Protein A ◽  
Yeast Cells ◽  
Type Ii ◽  
Ubiquitin System ◽  
Endoplasmic Reticulum Protein ◽  
Split Ubiquitin

The α1,2-mannosidase Mns1p involved in the N-glycosidic pathway in Saccharomyces cerevisiae is a type II membrane protein of the endoplasmic reticulum. The localization of Mns1p depends on retrieval from the Golgi through a mechanism that involves Rer1p. A chimera consisting of the transmembrane domain of Mns1p fused to the catalytic domain of the Golgi α1,2-mannosyltransferase Kre2p was localized in the endoplasmic reticulum of Δpep4 cells and in the vacuoles of rer1/Δpep4 by indirect immunofluorescence. The split-ubiquitin system was used to determine if there is an interaction between Mns1p and Rer1p in vivo. Co-expression of NubG-Mns1p and Rer1p-Cub-protein A-lexA-VP16 in L40 yeast cells resulted in cleavage of the reporter molecule, protein A-lexA-VP16, detected by western blot analysis and by expression of β-galactosidase activity. Sec12p, another endoplasmic reticulum protein that depends on Rer1p for its localization, also interacted with Rer1p using the split-ubiquitin assay, whereas the endoplasmic reticulum protein Ost1p showed no interaction. A weak interaction was observed between Alg5p and Rer1p. These results demonstrate that the transmembrane domain of Mns1p is sufficient for Rer1p-dependent endoplasmic reticulum localization and that Mns1p and Rer1p interact. Furthermore, the split-ubiquitin system demonstrates that the C-terminal of Rer1p is in the cytosol.

scholarly journals PKAc-directed interaction and phosphorylation of Ptc is required for Hh signaling inhibition in Drosophila

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Jialin Fan ◽  
Yajie Gao ◽  
Yi Lu ◽  
Wenqing Wu ◽  
Shuo Yuan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Kinase Activity ◽  
Functional Assays ◽  
Working Model ◽  
Inhibitory Function ◽  
Hh Signaling ◽  
Signaling Activation

Abstract Ptc is a gatekeeper to avoid abnormal Hh signaling activation, but the key regulators involved in Ptc-mediated inhibition remain largely unknown. Here, we identify PKAc as a key regulator required for Ptc inhibitory function. In the absence of Hh, PKAc physically interacts with Ptc and phosphorylates Ptc at Ser-1150 and -1183 residues. The presence of Hh unleashes PKAc from Ptc and activates Hh signaling. By combining both in vitro and in vivo functional assays, we demonstrate that such Ptc–PKAc interaction and Ptc phosphorylation are both important for Ptc inhibitory function. Interestingly, we further demonstrate that PKAc is subjected to palmitoylation, contributing to its kinase activity on plasma membrane. Based on those novel findings, we establish a working model on Ptc inhibitory function: In the absence of Hh, PKAc interacts with and phosphorylates Ptc to ensure its inhibitory function; and Hh presence releases PKAc from Ptc, resulting in Hh signaling activation.

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