Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins

Tirat, Aline; Freuler, Felix; Stettler, Thomas; Mayr, Lorenz M.; Leder, Lukas

doi:10.1016/j.ijbiomac.2006.01.012

Cited by 61 publications

(49 citation statements)

References 19 publications

(32 reference statements)

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“…SNAP is derived from alkyl guanine DNA alkyl transferase, a DNA repair enzyme that irreversibly transfers an alkyl group from its substrate to a reactive cysteine residue (32)(33)(34)(35)(36). The availability of cell-permeable nonfluorescent and fluorescent SNAP substrates, such as TMR-Star, allows for a broad variety of applications.…”

Section: Significancementioning

confidence: 99%

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies

Hoboth

Müller

Ivanova

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Insulin secretion is key for glucose homeostasis. Insulin secretory granules (SGs) exist in different functional pools, with young SGs being more mobile and preferentially secreted. However, the principles governing the mobility of age-distinct SGs remain undefined. Using the time-reporter insulin-SNAP to track age-distinct SGs we now show that their dynamics can be classified into three components: highly dynamic, restricted, and nearly immobile. Young SGs display all three components, whereas old SGs are either restricted or nearly immobile. Both glucose stimulation and F-actin depolymerization recruit a fraction of nearly immobile young, but not old, SGs for highly dynamic, microtubule-dependent transport. Moreover, F-actin marks multigranular bodies/lysosomes containing aged SGs. These data demonstrate that SGs lose their responsiveness to glucose stimulation and competence for microtubule-mediated transport over time while changing their relationship with F-actin.

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Section: Significancementioning

confidence: 99%

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies

Hoboth

Müller

Ivanova

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…To do this we constructed a dual-tagged Na,K-ATPase by fusing the SNAP tag at the N terminus of the HA-tagged Na,K-ATPase construct (10). The SNAP tag is a modified version of the DNA repair protein O 6 -alkylguanine-DNA alkyltransferase that can be covalently labeled with substituted groups presented as benzylguanine-based adducts (17,18). For this study, we utilized benzylguanine substituted with biotin (BG-biotin), such that, upon labeling, the SNAP tag becomes covalently biotinylated and amenable to standard streptavidin-biotin purification techniques.…”

Section: Resultsmentioning

confidence: 99%

Association with β-COP Regulates the Trafficking of the Newly Synthesized Na,K-ATPase*

Morton

Farr

Hull

et al. 2010

Journal of Biological Chemistry

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Plasma membrane expression of the Na,K-ATPase requires assembly of its ␣-and ␤-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase ␣-subunit and the coat protein, ␤-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase ␤-subunit, the Na,K-ATPase ␣-subunit interacts with ␤-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase ␤-subunit, the ␣-subunit does not interact with ␤-COP and traffics to the plasma membrane.Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase ␣-and ␤-subunits, newly synthesized ␣-subunit associates with ␤-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the ␣-and ␤-subunits to form the pump holoenzyme. The interaction with ␤-COP was reduced by mutating a dibasic motif at Lys 54 in the Na,K-ATPase ␣-subunit. This mutant ␣-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase ␤-subunit expression. Although the Lys 54 ␣-subunit reaches the cell surface without need for ␤-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the ␤-subunit.

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“…S1). To quantify this, we fused SMO N-terminal to an enzymatic self-labeling tag called SNAP that enables direct detection and quantification of the tagged protein after electrophoresis (Tirat et al, 2006). After checking the functionality of SNAP-SMO in vivo (Fig.…”

Section: δ978mentioning

confidence: 99%

Dose-dependent transduction of Hedgehog relies on phosphorylation-based feedback between the G-protein-coupled receptor Smoothened and the kinase Fused

et al. 2017

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Smoothened (SMO) is a G-protein-coupled receptor-related protein required for the transduction of Hedgehog (HH). The HH gradient leads to graded phosphorylation of SMO, mainly by the PKA and CKI kinases. How thresholds in HH morphogen regulate SMO to promote switch-like transcriptional responses is a central unsolved issue. Using the wing imaginal disc model in Drosophila, we identified new SMO phosphosites that enhance the effects of the PKA/CKI kinases on SMO accumulation, its localization at the plasma membrane and its activity. Surprisingly, phosphorylation at these sites is induced by the kinase Fused (FU), a known downstream effector of SMO. In turn, activation of SMO induces FU to act on its downstream targets. Overall, our data provide evidence for a SMO/FU positive regulatory loop nested within a multikinase phosphorylation cascade. We propose that this complex interplay amplifies signaling above a threshold that allows high HH signaling.

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Evaluation of two novel tag-based labelling technologies for site-specific modification of proteins

Cited by 61 publications

References 19 publications

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies

Association with β-COP Regulates the Trafficking of the Newly Synthesized Na,K-ATPase*

Dose-dependent transduction of Hedgehog relies on phosphorylation-based feedback between the G-protein-coupled receptor Smoothened and the kinase Fused

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