USP1 deubiquitinates Akt to inhibit PI3K-Akt-FoxO signaling in muscle during prolonged starvation.

PI3K-Akt-FoxO-mTOR signaling is the central pathway controlling growth and metabolism in all cells. Ubiquitination of the protein kinase Akt prior to its phosphorylation is required for PI3K-Akt activity. Here, we found that the deubiquitinating (DUB) enzyme USP1 removes K63-linked polyubiquitin chains on Akt to restrict PI3K-Akt-FoxO signaling in mouse muscle during prolonged starvation. DUB screening platform identified USP1 as a direct DUB for Akt, and USP1 depletion in mouse muscle increased Akt ubiquitination, PI3K-Akt-FoxO signaling, and glucose uptake during fasting. Co-immunoprecipitation and mass spectrometry identified disabled homolog-2 (Dab2), the tuberous sclerosis complex TSC1/TSC2, and PHLPP1 as USP1 bound proteins. During starvation, Dab2 is essential for Akt recruitment to USP1-TSC1-PHLPP1 complex, and for PI3K-Akt-FoxO inhibition. Surprisingly, USP1 limits TSC1 levels to sustain mTOR-mediated basal protein synthesis rates and maintain its own protein levels. We propose that Dab2 recruits Akt to USP1-TSC1-PHLPP1 complex to efficiently terminate the transmission of growth signals when cellular energy level is low.

A semiautomated measurement of muscle fiber size using the Imaris software.

The size and shape of skeletal muscle fibers are affected by various physiological and pathological conditions, such as muscle atrophy, hypertrophy, regeneration, and dystrophies. Hence, muscle fiber cross-sectional area (CSA) is an important determinant of muscle health and plasticity. We adapted the Imaris software to automatically segment muscle fibers based on fluorescent labeling of the plasma membrane and measure muscle fiber CSA. Analysis of muscle cross sections by the Imaris semiautomated and manual approaches demonstrated a similar decrease in CSA of atrophying muscles from fasted mice compared with fed controls. In addition, we previously demonstrated that downregulation of the Ca2+-specific protease calpain-1 attenuates muscle atrophy. Accordingly, both the Imaris semiautomated and manual approaches showed a similar increase in CSA of fibers expressing calpain-1 shRNA compared with adjacent nontransfected fibers in the same muscle cross section. Although both approaches seem valid for measurements of muscle fiber size, the manual marking method is less preferable because it is highly time-consuming, subjective, and limits the number of cells that can be analyzed. The Imaris semiautomated approach is user-friendly, requires little training or optimization, and can be used to efficiently and accurately mark thousands of fibers in a short period. As a novel addition to the commonly used statistics, we also describe statistical tests that quantify the strength of an effect on fiber size, enabling detection of significant differences between skewed distributions that would otherwise not be detected using typical methods.

Structure of the bovine COPI δ subunit µ homology domain at 2.15 Å resolution.

The heptameric COPI coat (coatomer) plays an essential role in vesicular transport in the early secretory system of eukaryotic cells. While the structures of some of the subunits have been determined, that of the δ-COP subunit has not been reported to date. The δ-COP subunit is part of a subcomplex with structural similarity to tetrameric clathrin adaptors (APs), where δ-COP is the structural homologue of the AP µ subunit. Here, the crystal structure of the µ homology domain (MHD) of δ-COP (δ-MHD) obtained by phasing using a combined SAD-MR method is presented at 2.15 Å resolution. The crystallographic asymmetric unit contains two monomers that exhibit short sections of disorder, which may allude to flexible regions of the protein. The δ-MHD is composed of two subdomains connected by unstructured linkers. Comparison between this structure and those of known MHD domains from the APs shows significant differences in the positions of specific loops and ß-sheets, as well as a more general change in the relative positions of the protein subdomains. The identified difference may be the major source of cargo-binding specificity. Finally, the crystal structure is used to analyze the potential effect of the I422T mutation in δ-COP previously reported to cause a neurodegenerative phenotype in mice.

Distinct role of subcomplexes of the COPI coat in the regulation of ArfGAP2 activity.

COPI vesicles serve for transport of proteins and membrane lipids in the early secretory pathway. Their coat protein (coatomer) is a heptameric complex that is recruited to the Golgi by the small GTPase Arf1. Although recruited en bloc, coatomer can be viewed as a stable assembly of an adaptin-like tetrameric subcomplex (CM4) and a trimeric 'cage' subcomplex (CM3). Following recruitment, coatomer stimulates ArfGAP-dependent GTP hydrolysis on Arf1. Here, we employed recombinant coatomer subcomplexes to study the role of coatomer components in the regulation of ArfGAP2, an ArfGAP whose activity is strictly coatomer-dependent. Within CM4, we define a novel hydrophobic pocket for ArfGAP2 interaction on the appendage domain of γ1-COP. The CM4 subcomplex (but not CM3) is recruited to membranes through Arf1 and can subsequently recruit ArfGAP2. Neither CM3 nor CM4 in itself is effective in stimulating ArfGAP2 activity, but stimulation is regained when both subcomplexes are present. Our findings point to a distinct role of each of the two coatomer subcomplexes in the regulation of ArfGAP2-dependent GTP hydrolysis on Arf1, where the CM4 subcomplex functions in GAP recruitment, while, similarly to the COPII system, the cage-like CM3 subcomplex stimulates the catalytic reaction.

ArfGAP1 interacts with coat proteins through tryptophan-based motifs.

The Arf1 GTPase-activating protein ArfGAP1 regulates vesicular traffic through the COPI system. This protein consists of N-terminal catalytic domain, lipid packing sensors (the ALPS motifs) in the central region, and a carboxy part of unknown function. The carboxy part contains several diaromatic sequences that are reminiscent of motifs known to interact with clathrin adaptors. In pull-down experiments using GST-fused peptides from rat ArfGAP1, a peptide containing a (329)WETF sequence interacted strongly with clathrin adaptors AP1 and AP2, whereas a major coatomer-binding determinant was identified within the extreme carboxy terminal peptide ((405)AADEGWDNQNW). Mutagenesis and peptide competition experiments revealed that this determinant is required for coatomer binding to full-length ArfGAP1, and that interaction is mediated through the delta-subunit of the coatomer adaptor-like subcomplex. Evidence for a role of the carboxy motif in ArfGAP1-coatomer interaction in vivo is provided by means of a reporter fusion assay. Our findings point to mechanistic differences between ArfGAP1 and the other ArfGAPs known to function in the COPI system.

A signaling hub of insulin receptor, dystrophin glycoprotein complex and plakoglobin regulates muscle size.

Signaling through the insulin receptor governs central physiological functions related to cell growth and metabolism. Here we show by tandem native protein complex purification approach and super-resolution STED microscopy that insulin receptor activity requires association with the fundamental structural module in muscle, the dystrophin glycoprotein complex (DGC), and the desmosomal component plakoglobin (γ-catenin). The integrity of this high-molecular-mass assembly renders skeletal muscle susceptibility to insulin, because DGC-insulin receptor dissociation by plakoglobin downregulation reduces insulin signaling and causes atrophy. Furthermore, low insulin receptor activity in muscles from transgenic or fasted mice decreases plakoglobin-DGC-insulin receptor content on the plasma membrane, but not when plakoglobin is overexpressed. By masking ß-dystroglycan LIR domains, plakoglobin prevents autophagic clearance of plakoglobin-DGC-insulin receptor co-assemblies and maintains their function. Our findings establish DGC as a signaling hub, and provide a possible mechanism for the insulin resistance in Duchenne Muscular Dystrophy, and for the cardiomyopathies seen with plakoglobin mutations.

High abundance of ArfGAP1 found in the mossy fibers in hilus of the dentate gyrus region of the mouse brain.

The Arf GTPase-activating protein ArfGAP1 and its brain-specific isoform ArfGAP1B play an important role in neurotransmission. Here we analyzed the distribution of ArfGAP1 in the mouse brain. We found high levels of ArfGAP1 in the mouse dentate gyrus where it displayed especially elevated level in the polymorph layer (hilus). Importantly, the ArfGAP1 signal follows the pathway of the granular cell axons so-called mossy fibers which extend from the dentate gyrus to CA3 via stratum lucidum and partially stratum oriens. Additionally, we identified differential expression of ArfGAP1 in the isocortex. Thus, staining with anti-ArfGAP1 antibodies allows distinction between cortical cell layers 1, 2, 3 and 5 from 4 and 6. Taken together, our data suggest that ArfGAP1 can be used as a specific marker of the dentate mossy fibers and as for visualization of cortical layers in immunohistochemical studies.

Control of protein trafficking by reversible masking of transport signals.

Systems that allow the control of protein traffic between subcellular compartments have been valuable in elucidating trafficking mechanisms. Most current approaches rely on ligand or light-controlled dimerization, which results in either retardation or enhancement of the transport of a reporter. We developed an alternative approach for trafficking regulation that we term "controlled unmasking of targeting elements" (CUTE). Regulated trafficking is achieved by reversible masking of the signal that directs the reporter to its target organelle, relying on the streptavidin-biotin system. The targeting signal is generated within or immediately after a 38-amino acid streptavidin-binding peptide (SBP) that is appended to the reporter. The binding of coexpressed streptavidin to SBP causes signal masking, whereas addition of biotin causes complex dissociation and triggers protein transport to the target organelle. We demonstrate the application of this approach to the control of nuclear and peroxisomal protein import and the generation of biotin-dependent trafficking through the endocytic and COPI systems. By simultaneous masking of COPI and endocytic signals, we were able to generate a synthetic pathway for efficient transport of a reporter from the plasma membrane to the endoplasmic reticulum.

Discrete determinants in ArfGAP2/3 conferring Golgi localization and regulation by the COPI coat.

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein-protein and protein-lipid interactions to promote GTP hydrolysis in Arf1-GTP.

Topology of amphipathic motifs mediating Golgi localization in ArfGAP1 and its splice isoforms.

The interaction of the Arf1-directed GTPase-activating protein ArfGAP1 with the Golgi apparatus depends on motifs in its noncatalytic part that are unstructured in solution but are capable of folding into amphipathic helices in vitro upon interaction with poorly packed lipids. In previous studies a few hydrophobic residues that are critical for lipid binding and Golgi localization were identified, but the precise topology of the amphipathic motifs has not been determined. Here we present a detailed analysis of the Golgi targeting and in vitro folding features of the region encompassing the amphipathic motifs (residues 199-294). Point mutation analysis revealed that most hydrophobic residues within this region contribute to Golgi localization, whereas analysis by proline replacements and alanine insertions revealed that Golgi interaction depends on folding into two amphipathic helices with a short interrupting sequence. Analysis of splice isoforms containing 10-residue in-frame insertions within their first amphipathic motifs revealed that the insertion causes a truncation of the amphipathic helix that does not extend beyond the insertion sequence. Lastly, a lysine replacement mutant recently reported to bind to negatively charged liposomes in a curvature-independent manner showed normal cellular distribution, suggesting that Golgi targeting of Arf-GAP1 may involve factors other than sensing lipid packing.

Golgi localization determinants in ArfGAP1 and in new tissue-specific ArfGAP1 isoforms.

The Arf1-directed GTPase-activating protein ArfGAP1 is a Golgi-localized protein that controls the dynamics of the COPI coat of carriers that mediate transport in the endoplasmic reticulum-Golgi shuttle. Previously the interaction of ArfGAP1 with the Golgi was allocated to a portion of the non-catalytic, carboxyl part of the protein, but the mechanism of this interaction has not been established. In this study we identify a short stretch in the non-catalytic part of ArfGAP1 (residues 204-214) in which several hydrophobic residues contribute to Golgi localization. Even single alanine replacement of two of these residues (Leu-207 and Trp-211) strongly diminished Golgi localization. Mutations in the hydrophobic residues also diminished the in vitro activity of ArfGAP1 on Arf1 bound to Golgi membranes. The stretch containing the hydrophobic residues was recently shown to mediate the binding of ArfGAP1 to loosely packed lipids of highly curved liposomes (Bigay, J., Casella, J. F., Drin, G., Mesmin, B., and Antonny, B. (2005) EMBO J. 24, 2244-2253). Whereas short fragments containing the hydrophobic stretch were not Golgi-localized, a proximal 10-residue in-frame insertion that is present in new ArfGAP1 isoforms that we identified in brain and heart tissues could confer Golgi localization on these fragments. This localization was abrogated by alanine replacement of residues Phe-240 or Trp-241 of the insertion sequence but not by their replacement with leucines. Our findings indicate that ArfGAP1 interacts with the Golgi through multiple hydrophobic motifs and that alternative modes of interaction may exist in tissue-specific ArfGAP1 isoforms.

The CCAAT binding factor can mediate interactions between CONSTANS-like proteins and DNA.

CONSTANS-Like (COL) proteins are plant-specific nuclear regulators of gene expression but do not contain a known DNA-binding motif. We tested whether a common DNA-binding protein can deliver these proteins to specific cis-acting elements. We screened for proteins that interact with two members of a subgroup of COL proteins. These COL proteins were Tomato COL1 (TCOL1), which does not seem to be involved in the control of flowering time, and the Arabidopsis thaliana CONSTANS (AtCO) protein which mediates photoperiodic induction of flowering. We show that the C-terminal plant-specific CCT (CO, CO-like, TIMING OF CAB EXPRESSION 1) domain of both proteins binds the trimeric CCAAT binding factor (CBF) via its HAP5/NF-YC component. Chromatin immunoprecipitation demonstrated that TCOL is recruited to the CCAAT motifs of the yeast CYC1 and HEM1 promoters by HAP5. In Arabidopsis, each of the three CBF components is encoded by several different genes that are highly transcribed. Under warm long days, high levels of expression of a tomato HAP5 (THAP5a) gene can reduce the flowering time of Arabidopsis. A mutation in the CCT domain of TCOL1 disrupts the interaction with THAP5 and the analogous mutation in AtCO impairs its function and delays flowering. CBFs are therefore likely to recruit COL proteins to their DNA target motifs in planta.