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LSSS 2017-2018


Life Sciences Seminar Series


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Blanche Schwappach

Universitätsmedizin Göttingen, Germany Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany

The GET/TRC40 pathway at the intersection of membrane protein targeting and proteostasis

Talk abstract

The group works on different aspects of membrane protein biogenesis and its integration into the physiology of organs such as the brain or the heart. We study the early life of tail-anchored proteins that are post-translationally targeted to the endoplasmic reticulum for membrane integration. Other projects address the role of sorting motifs during the passage of ion channels and neurotransmitter receptors through the secretory pathway. One channel under investigation (the KATP channel) couples cellular metabolism to insulin secretion in pancreatic beta cells. In the brain and the heart KATP channels play less defined roles that we currently address employing biochemical methods. We study biogenesis and trafficking under (patho)physiological conditions in genetically tractable model organisms such as yeast or mouse. Besides membrane protein biochemistry we use GFP-based physiological sensors for small molecules and ions in cellular compartments. This allows us to tackle how ion channels and transporters contribute to different physicochemical milieus inside cells.

Selected Publications

Two novel effectors of trafficking and maturation of the yeast plasma membrane H+ -ATPase.Geva Y, Crissman J, Arakel EC, Gómez-Navarro N, Chuartzman SG, Stahmer KR, Schwappach B, Miller EA, Schuldiner M
Traffic 2017 Oct; 18(10):672-682


The endoplasmic reticulum (ER) is the entry site of proteins into the endomembrane system. Proteins exit the ER via coat protein II (COPII) vesicles in a selective manner, mediated either by direct interaction with the COPII coat or aided by cargo receptors. Despite the fundamental role of such receptors in protein sorting, only a few have been identified. To further define the machinery that packages secretory cargo and targets proteins from the ER to Golgi membranes, we used multiple systematic approaches, which revealed 2 uncharacterized proteins that mediate the trafficking and maturation of Pma1, the essential yeast plasma membrane proton ATPase. Ydl121c (Exp1) is an ER protein that binds Pma1, is packaged into COPII vesicles, and whose deletion causes ER retention of Pma1. Ykl077w (Psg1) physically interacts with Exp1 and can be found in the Golgi and coat protein I (COPI) vesicles but does not directly bind Pma1. Loss of Psg1 causes enhanced degradation of Pma1 in the vacuole. Our findings suggest that Exp1 is a Pma1 cargo receptor and that Psg1 aids Pma1 maturation in the Golgi or affects its retrieval. More generally our work shows the utility of high content screens in the identification of novel trafficking components.

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function.Arakel EC, Richter KP, Clancy A, Schwappach B
Proc Natl Acad Sci U S A 2016 Jun 21; 113(25):6916-21


Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer.

A dual phosphorylation switch controls 14-3-3-dependent cell surface expression of TASK-1.Kilisch M, Lytovchenko O, Arakel EC, Bertinetti D, Schwappach B
J Cell Sci 2016 Feb 15; 129(4):831-42


The transport of the K(+) channels TASK-1 and TASK-3 (also known as KCNK3 and KCNK9, respectively) to the cell surface is controlled by the binding of 14-3-3 proteins to a trafficking control region at the extreme C-terminus of the channels. The current model proposes that phosphorylation-dependent binding of 14-3-3 sterically masks a COPI-binding motif. However, the direct effects of phosphorylation on COPI binding and on the binding parameters of 14-3-3 isoforms are still unknown. We find that phosphorylation of the trafficking control region prevents COPI binding even in the absence of 14-3-3, and we present a quantitative analysis of the binding of all human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-3. Surprisingly, the affinities of 14-3-3 proteins for TASK-1 are two orders of magnitude lower than for TASK-3. Furthermore, we find that phosphorylation of a second serine residue in the C-terminus of TASK-1 inhibits 14-3-3 binding. Thus, phosphorylation of the trafficking control region can stimulate or inhibit transport of TASK-1 to the cell surface depending on the target serine residue. Our findings indicate that control of TASK-1 trafficking by COPI, kinases, phosphatases and 14-3-3 proteins is highly dynamic.

The laboratory notebook in the 21st century: The electronic laboratory notebook would enhance good scientific practice and increase research productivity.Nussbeck SY, Weil P, Menzel J, Marzec B, Lorberg K, Schwappach B
EMBO Rep 2014 Jun; 15(6):631-4


The yeast CLC protein counteracts vesicular acidification during iron starvation.Braun NA, Morgan B, Dick TP, Schwappach B
J Cell Sci 2010 Jul 1; 123(Pt 13):2342-50


Ion gradients across intracellular membranes contribute to the physicochemical environment inside compartments. CLC anion transport proteins that localise to intracellular organelles are anion-proton exchangers involved in anion sequestration or vesicular acidification. By homology, the only CLC protein of Saccharomyces cerevisiae, Gef1, belongs to this family of intracellular exchangers. Gef1 localises to the late Golgi and prevacuole and is essential in conditions of iron limitation. In the absence of Gef1, a multicopper oxidase involved in iron uptake, Fet3, fails to acquire copper ion cofactors. The precise role of the exchanger in this physiological context is unknown. Here, we show that the Gef1-containing compartment is adjusted to a more alkaline pH under iron limitation. This depends on the antiport function of Gef1, because an uncoupled mutant of Gef1 (E230A) results in the acidification of the lumen and fails to support Fet3 maturation. Furthermore, we found that Gef1 antiport activity correlates with marked effects on cellular glutathione homeostasis, raising the possibility that the effect of Gef1 on Fet3 copper loading is related to the control of compartmental glutathione concentration or redox status. Mutational inactivation of a conserved ATP-binding site in the cytosolic cystathione beta-synthetase domain of Gef1 (D732A) suggests that Gef1 activity is regulated by energy metabolism.