Complex consequences of Cantu syndrome SUR2 variant R1154Q in genetically modified mice.

Cantu syndrome (CS) is caused by gain-of-function (GOF) mutations in pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits, the most common mutations being SUR2[R1154Q] and SUR2[R1154W], carried by approximately 30% of patients. We used CRISPR/Cas9 genome engineering to introduce the equivalent of the human SUR2[R1154Q] mutation into the mouse ABCC9 gene. Along with minimal CS disease features, R1154Q cardiomyocytes and vascular smooth muscle showed much lower KATP current density and pinacidil activation than WT cells. Almost complete loss of SUR2-dependent protein and KATP in homozygous R1154Q ventricles revealed underlying diazoxide-sensitive SUR1-dependent KATP channel activity. Surprisingly, sequencing of SUR2 cDNA revealed 2 distinct transcripts, one encoding full-length SUR2 protein; and the other with an in-frame deletion of 93 bases (corresponding to 31 amino acids encoded by exon 28) that was present in approximately 40% and approximately 90% of transcripts from hetero- and homozygous R1154Q tissues, respectively. Recombinant expression of SUR2A protein lacking exon 28 resulted in nonfunctional channels. CS tissue from SUR2[R1154Q] mice and human induced pluripotent stem cell-derived (hiPSC-derived) cardiomyocytes showed only full-length SUR2 transcripts, although further studies will be required in order to fully test whether SUR2[R1154Q] or other CS mutations might result in aberrant splicing and variable expressivity of disease features in human CS.

Dissection of GTPase-activating proteins reveals functional asymmetry in the COPI coat of budding yeast.

The Arf GTPase controls formation of the COPI vesicle coat. Recent structural models of COPI revealed the positioning of two Arf1 molecules in contrasting molecular environments. Each of these pockets for Arf1 is expected to also accommodate an Arf GTPase-activating protein (ArfGAP). Structural evidence and protein interactions observed between isolated domains indirectly suggest that each niche preferentially recruits one of the two ArfGAPs known to affect COPI, i.e. Gcs1/ArfGAP1 and Glo3/ArfGAP2/3, although only partial structures are available. The functional role of the unique non-catalytic domain of either ArfGAP has not been integrated into the current COPI structural model. Here, we delineate key differences in the consequences of triggering GTP hydrolysis through the activity of one versus the other ArfGAP. We demonstrate that Glo3/ArfGAP2/3 specifically triggers Arf1 GTP hydrolysis impinging on the stability of the COPI coat. We show that the Snf1 kinase complex, the yeast homologue of AMP-activated protein kinase (AMPK), phosphorylates the region of Glo3 that is crucial for this effect and, thereby, regulates its function in the COPI-vesicle cycle. Our results revise the model of ArfGAP function in the molecular context of COPI.This article has an associated First Person interview with the first author of the paper.

Toolbox: Creating a systematic database of secretory pathway proteins uncovers new cargo for COPI.

A third of yeast genes encode for proteins that function in the endomembrane system. However, the precise localization for many of these proteins is still uncertain. Here, we visualized a collection of ~500 N-terminally, green fluorescent protein (GFP), tagged proteins of the yeast Saccharomyces cerevisiae. By co-localizing them with 7 known markers of endomembrane compartments we determined the localization for over 200 of them. Using this approach, we create a systematic database of the various secretory compartments and identify several new residents. Focusing in, we now suggest that Lam5 resides in contact sites between the endoplasmic reticulum and the late Golgi. Additionally, analysis of interactions between the COPI coat and co-localizing proteins from our screen identifies a subset of proteins that are COPI-cargo. In summary, our approach defines the protein roster within each compartment enabling characterization of the physical and functional organization of the endomembrane system and its components.

Formation of COPI-coated vesicles at a glance.

The coat protein complex I (COPI) allows the precise sorting of lipids and proteins between Golgi cisternae and retrieval from the Golgi to the ER. This essential role maintains the identity of the early secretory pathway and impinges on key cellular processes, such as protein quality control. In this Cell Science at a Glance and accompanying poster, we illustrate the different stages of COPI-coated vesicle formation and revisit decades of research in the context of recent advances in the elucidation of COPI coat structure. By calling attention to an array of questions that have remained unresolved, this review attempts to refocus the perspectives of the field.

Looking inside the cell.

Advances in imaging techniques have shed new light on the structure of vesicles formed by COPI protein complexes.

Two novel effectors of trafficking and maturation of the yeast plasma membrane H+ -ATPase.

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.

The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.

In eukaryotes, up to one-third of cellular proteins are targeted to the endoplasmic reticulum, where they undergo folding, processing, sorting and trafficking to subsequent endomembrane compartments. Targeting to the endoplasmic reticulum has been shown to occur co-translationally by the signal recognition particle (SRP) pathway or post-translationally by the mammalian transmembrane recognition complex of 40 kDa (TRC40) and homologous yeast guided entry of tail-anchored proteins (GET) pathways. Despite the range of proteins that can be catered for by these two pathways, many proteins are still known to be independent of both SRP and GET, so there seems to be a critical need for an additional dedicated pathway for endoplasmic reticulum relay. We set out to uncover additional targeting proteins using unbiased high-content screening approaches. To this end, we performed a systematic visual screen using the yeast Saccharomyces cerevisiae, and uncovered three uncharacterized proteins whose loss affected targeting. We suggest that these proteins work together and demonstrate that they function in parallel with SRP and GET to target a broad range of substrates to the endoplasmic reticulum. The three proteins, which we name Snd1, Snd2 and Snd3 (for SRP-independent targeting), can synthetically compensate for the loss of both the SRP and GET pathways, and act as a backup targeting system. This explains why it has previously been difficult to demonstrate complete loss of targeting for some substrates. Our discovery thus puts in place an essential piece of the endoplasmic reticulum targeting puzzle, highlighting how the targeting apparatus of the eukaryotic cell is robust, interlinked and flexible.

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function.

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.

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 molecular and functional identities of atrial cardiomyocytes in health and disease.

Atrial cardiomyocytes are essential for fluid homeostasis, ventricular filling, and survival, yet their cell biology and physiology are incompletely understood. It has become clear that the cell fate of atrial cardiomyocytes depends significantly on transcription programs that might control thousands of differentially expressed genes. Atrial muscle membranes propagate action potentials and activate myofilament force generation, producing overall faster contractions than ventricular muscles. While atria-specific excitation and contractility depend critically on intracellular Ca(2+) signalling, voltage-dependent L-type Ca(2+) channels and ryanodine receptor Ca(2+) release channels are each expressed at high levels similar to ventricles. However, intracellular Ca(2+) transients in atrial cardiomyocytes are markedly heterogeneous and fundamentally different from ventricular cardiomyocytes. In addition, differential atria-specific K(+) channel expression and trafficking confer unique electrophysiological and metabolic properties. Because diseased atria have the propensity to perpetuate fast arrhythmias, we discuss our understanding about the cell-specific mechanisms that lead to metabolic and/or mitochondrial dysfunction in atrial fibrillation. Interestingly, recent work identified potential atria-specific mechanisms that lead to early contractile dysfunction and metabolic remodelling, suggesting highly interdependent metabolic, electrical, and contractile pathomechanisms. Hence, the objective of this review is to provide an integrated model of atrial cardiomyocytes, from tissue-specific cell properties, intracellular metabolism, and excitation-contraction (EC) coupling to early pathological changes, in particular metabolic dysfunction and tissue remodelling due to atrial fibrillation and aging. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

Tuning the electrical properties of the heart by differential trafficking of KATP ion channel complexes.

The copy number of membrane proteins at the cell surface is tightly regulated. Many ion channels and receptors present retrieval motifs to COPI vesicle coats and are retained in the early secretory pathway. In some cases, the interaction with COPI is prevented by binding to 14-3-3 proteins. However, the functional significance of this antagonism between COPI and 14-3-3 in terminally differentiated cells is unknown. Here, we show that ATP-sensitive K(+) (KATP) channels, which are composed of Kir6.2 and SUR1 subunits, are stalled in the Golgi complex of ventricular, but not atrial, cardiomyocytes. Upon sustained ß-adrenergic stimulation, which leads to activation of protein kinase A (PKA), SUR1-containing channels reach the plasma membrane of ventricular cells. We show that PKA-dependent phosphorylation of the C-terminus of Kir6.2 decreases binding to COPI and, thereby, silences the arginine-based retrieval signal. Thus, activation of the sympathetic nervous system releases this population of KATP channels from storage in the Golgi and, hence, might facilitate the adaptive response to metabolic challenges.