Split-Ubiquitin and the Split-Protein Sensors: Chessman for the Endgame

ABSTRACT Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1 Δ hph2 Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71 Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1 Δ hph2 Δ and hph1 Δ hph2 Δ sec71 Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1 Δ hph2 Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

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

Journal of Cell Science ◽

10.1242/jcs.114.24.4629 ◽

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.

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The split protein phosphatase system

Biochemical Journal ◽

10.1042/bcj20170726 ◽

2018 ◽

Vol 475 (23) ◽

pp. 3707-3723 ◽

Cited By ~ 9

Author(s):

Anne Bertolotti

Keyword(s):

Protein Phosphatase ◽

Catalytic Subunit ◽

Regulatory Subunit ◽

Post Translational Modification ◽

Antagonistic Action ◽

Cellular Processes ◽

Depth Study ◽

Split Protein ◽

Phosphatase System ◽

Physiological Substrates

Reversible phosphorylation of proteins is a post-translational modification that regulates all aspect of life through the antagonistic action of kinases and phosphatases. Protein kinases are well characterized, but protein phosphatases have been relatively neglected. Protein phosphatase 1 (PP1) catalyzes the dephosphorylation of a major fraction of phospho-serines and phospho-threonines in cells and thereby controls a broad range of cellular processes. In this review, I will discuss how phosphatases were discovered, how the view that they were unselective emerged and how recent findings have revealed their exquisite selectivity. Unlike kinases, PP1 phosphatases are obligatory heteromers composed of a catalytic subunit bound to one (or two) non-catalytic subunit(s). Based on an in-depth study of two holophosphatases, I propose the following: selective dephosphorylation depends on the assembly of two components, the catalytic subunit and the non-catalytic subunit, which serves as a high-affinity substrate receptor. Because functional complementation of the two modules is required to produce a selective holophosphatase, one can consider that they are split enzymes. The non-catalytic subunit was often referred to as a regulatory subunit, but it is, in fact, an essential component of the holoenzyme. In this model, a phosphatase and its array of mostly orphan substrate receptors constitute the split protein phosphatase system. The set of potentially generalizable principles outlined in this review may facilitate the study of these poorly understood enzymes and the identification of their physiological substrates.

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Human PIEZO1 Ion Channel Functions as a Split Protein

PLoS ONE ◽

10.1371/journal.pone.0151289 ◽

2016 ◽

Vol 11 (3) ◽

pp. e0151289 ◽

Cited By ~ 7

Author(s):

Chilman Bae ◽

Thomas M. Suchyna ◽

Lynn Ziegler ◽

Frederick Sachs ◽

Philip A. Gottlieb

Keyword(s):

Ion Channel ◽

Split Protein

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β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides

Biomolecules ◽

10.3390/biom8030051 ◽

2018 ◽

Vol 8 (3) ◽

pp. 51 ◽

Cited By ~ 5

Author(s):

Shane Stone ◽

Tatjana Heinrich ◽

Suzy Juraja ◽

Jiulia Satiaputra ◽

Clinton Hall ◽

...

Keyword(s):

Functional Characterization ◽

Cell Penetrating Peptides ◽

Stable Cell Line ◽

Intracellular Space ◽

Biologically Relevant ◽

Cell Internalization ◽

Cytosolic Delivery ◽

Cell Penetrating ◽

Split Protein

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

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