AP-4 vesicles contribute to spatial control of autophagy via RUSC-dependent peripheral delivery of ATG9A.

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including 'Dynamic Organellar Maps', to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the trans-Golgi network to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the "ATG9A reservoir" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy

Mechanisms and dynamics of the NH2+_2^{+} + H+^{+} and NH+^{+} + H+^{+} + H fragmentation channels upon single-photon double ionization of NH3_3

We present state-selective measurements on the NH$_2^{+}$ + H$^{+}$ and NH$^{+}$ + H$^{+}$ + H dissociation channels following single-photon double ionization at 61.5 eV of neutral NH$_{3}$, where the two photoelectrons and two cations are measured in coincidence using 3-D momentum imaging. Three dication electronic states are identified to contribute to the NH$_2^{+}$ + H$^{+}$ dissociation channel, where the excitation in one of the three states undergoes intersystem crossing prior to dissociation, producing a cold NH$_2^+$ fragment. In contrast, the other two states directly dissociate, producing a ro-vibrationally excited NH$_2^+$ fragment with roughly 1 eV of internal energy. The NH$^{+}$ + H$^{+}$ + H channel is fed by direct dissociation from three intermediate dication states, one of which is shared with the NH$_2^{+}$ + H$^{+}$ channel. We find evidence of autoionization contributing to each of the double ionization channels. The distributions of the relative emission angle between the two photoelectrons, as well as the relative angle between the recoil axis of the molecular breakup and the polarization vector of the ionizing field, are also presented to provide insight on both the photoionization and photodissociation mechanisms for the different dication states.Comment: 18 pages, 21 figures, 3 table

Photoelectron and fragmentation dynamics of the H+^{+} + H+^{+} dissociative channel in NH3_3 following direct single-photon double ionization

We report measurements on the H$^{+}$ + H$^{+}$ fragmentation channel following direct single-photon double ionization of neutral NH$_{3}$ at 61.5 eV, where the two photoelectrons and two protons are measured in coincidence using 3-D momentum imaging. We identify four dication electronic states that contribute to H$^{+}$ + H$^{+}$ dissociation, based on our multireference configuration-interaction calculations of the dication potential energy surfaces. Of these four dication electronic states, three dissociate in a concerted process, while the fourth undergoes a sequential fragmentation mechanism. We find evidence that the neutral NH fragment or intermediate NH$^+$ ion is markedly ro-vibrationally excited. We also identify differences in the relative emission angle between the two photoelectrons as a function of their energy sharing for the four different dication states, which bare some similarities to previous observations made on atomic targets.Comment: 15 pages, 13 figures, 3 table

Region and cell-type resolved quantitative proteomic map of the human heart

The heart is a central human organ and its diseases are the leading cause of death worldwide, but an in-depth knowledge of the identity and quantity of its constituent proteins is still lacking. Here, we determine the healthy human heart proteome by measuring 16 anatomical regions and three major cardiac cell types by high-resolution mass spectrometry-based proteomics. From low microgram sample amounts, we quantify over 10,700 proteins in this high dynamic range tissue. We combine copy numbers per cell with protein organellar assignments to build a model of the heart proteome at the subcellular level. Analysis of cardiac fibroblasts identifies cellular receptors as potential cell surface markers. Application of our heart map to atrial fibrillation reveals individually distinct mitochondrial dysfunctions. The heart map is available at maxqb. biochem. mpg. de as a resource for future analyses of normal heart function and disease

SILAC-based quantitative mass spectrometry-based proteomics quantifies endoplasmic reticulum stress in whole HeLa cells

The unfolded protein response (UPR) involves extensive proteome remodeling in many cellular compartments. So far, a comprehensive analysis has been missing due to technological limitations. Here we employ Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based proteomics to quantify over 6200 proteins at increasing concentrations of tunicamycin in HeLa cells. We further compare the effects of tunicamycin (5 ug/ml) to those of thapsigargin (1 \ub5M) and DTT (2mM), both activating the UPR through different mechanisms. The systematic quantification of the proteome-wide expression changes following proteostatic stress is a resource for the scientific community, which enables the discovery of novel players involved in the pathophysiology of the broad range of disorders linked to proteostasis. We identified 38 proteins not previously linked to the UPR, whose expression increases, of which 15 likely remediate ER stress, and the remainder may contribute to pathological outcomes. Unexpectedly, there are few strongly downregulated proteins, despite expression of the pro-apoptotic transcription factor CHOP, suggesting that IRE1-dependent mRNA decay (RIDD) has a limited contribution to ER-stress mediated cell death in our system

AP-5 knockout affects Golgi-localised proteins.

<p>(A) Scatterplot comparing proteins that were depleted in vesicle-enriched fractions from SILAC-labelled AP5Z1_KO1 and AP5Z1_KO2 cells. Although AP-5 was not detected in the vesicle-enriched fraction, we looked for proteins whose trafficking through vesicular carriers might be altered by the loss of AP-5. Results are based on 2 biological repeats and show 5 Golgi-associated transmembrane proteins that were consistently depleted in both independent AP5Z1 knockouts. Data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.s004" target="_blank">S2 Data</a>. (B) Schematic diagram showing the topologies of the Golgi-associated proteins that were identified as possible hits in Fig 5A. (C) Indirect immunofluorescence microscopy of GOLIM4, GLG1, GALNT2, and GOLM1. The addition of manganese (Mn²⁺) for 4 h results in the redistribution of both GOLIM4 and GLG1 away from the Golgi region of the cell. Scale bar: 20 μm. (D) Whole cell lysates from control and Mn²⁺-treated cells. The addition of Mn²⁺ reduces the GOLIM4 signal by 52% and the GLG1 signal by 89%, presumably due to lysosomal degradation and consistent with the diminished immunofluorescence signal. AP, adaptor protein; CHC, clathrin heavy chain; con, control; KO, knockout; Mn²⁺, manganese; SILAC, stable isotope labelling of amino acids in cell culture.</p

CIMPR trafficking in control and AP5Z1 knockout cells.

<p>(A) Schematic representation of the CIMPR retrieval assay, which follows the trafficking of endogenous CIMPR. (B) Indirect immunofluorescence microscopy of control and AP5Z1 knockout cells, which were pulse chased with an antibody against CIMPR, as shown in Fig 3A. In both knockout lines, there was reduced retrieval of anti-CIMPR back to the juxtanuclear region, as defined by TGN46 labelling. These results are consistent with the identification of AP-5 ζ (KIAA0415) as a potential hit in a genome-wide screen for proteins involved in endosomal retrieval [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.ref017" target="_blank">17</a>]. Scale bar: 20 μm. (C) Quantification of the retrieval defect using an Arrayscan automated microscope. The whole cell stain allowed a mask to be drawn around the cells (red), an offset line was added to ensure that the whole cell was captured (blue), and the anti-TGN46 allowed a mask to be drawn around the TGN (pink). The CIMPR that failed to be retrieved back to the TGN is shown in yellow. Scale bar: 20 μm. (D) Analysis of the data from the Arrayscan microscope, using a Colocalisation Bioapplication. The fold increase in CIMPR (Object Total Area) that failed to be retrieved back to the juxtanuclear was 1.55 ± 0.04 for AP5Z1_KO1 and 1.32 ± 0.04 for AP5Z1_KO2. More than 1,500 cells were scored per knockdown condition (4 independent repeats; error bars indicate SEM). The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.s006" target="_blank">S4 Data</a>. Ab, antibody; AP, adaptor protein; CIMPR, cation-independent mannose 6-phosphate receptor; con, control; KO, knockout; TGN, trans-Golgi network; WCS, whole cell stain.</p

Effect of combined loss of retromer and AP-5 on CIMPR retrieval.

<p>(A) Immunofluorescence microscopy of control or AP5Z1 knockout cells depleted of VPS35 and pulse chased with anti-CIMPR, as shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.g003" target="_blank">Fig 3A</a>. Individually, the knockout of AP5Z1 or the knockdown of retromer caused a reduction in the retrieval of CIMPR back to the TGN region; this was further exacerbated when the knockout and knockdown were combined. The dotted lines indicate the boundaries of each cell. Scale bar: 20 μm. (B) Quantification of the retrieval defect of CIMPR, using the CX7 automated microscope and a Colocalisation Bioapplication. The increase in CIMPR (Total Object Count) that failed to be retrieved back to the TGN region was 1.48 ± 0.10 for VPS35 kd, 1.26 ± 0.07 for KO1, and 1.97 ± 0.14 for the combined KO1 plus VPS35 kd. More than 1,500 cells were scored per knockdown condition (7 independent repeats; error bars indicate SEM). The raw data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.s006" target="_blank">S4 Data</a>. (C) Whole cell lysates from control and AP5Z1 knockout lines. The steady-state levels of CIMPR are not significantly affected by loss of AP-5 or depletion of VPS35. In addition, the surface expression of CIMPR did not change substantially in the knockdown and knockout cells when compared to control, as determined by flow cytometry (VPS35kd 1.22 ± 0.36, KO1 1.12 ± 0.18, KO1+VPS35kd 1.25 ± 0.44). AP, adaptor protein; CHC, clathrin heavy chain; CIMPR, cation-independent mannose 6-phosphate receptor; con, control; KO, knockout; TGN, trans-Golgi network.</p

Comparison of control and AP5Z1-deficient cells.

<p>(A) Whole cell lysates from control HeLa cells and HeLa AP5Z1 knockout lines (KO1 and KO2) (first three lanes), and control and AP5Z1 patient-derived fibroblast lines AP5Z1*_1 p.(Q587*) and AP5Z1* p.(R138*);(p.W441*) [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.ref009" target="_blank">9</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.ref015" target="_blank">15</a>] (second three lanes). In the knockout and patient lines, there is complete loss of AP-5 ζ. CHC was included as a loading control. (B) Global proteome analysis. Left panel, HeLa cells: AP5Z1 knockout versus controls. Right panel, human fibroblasts: AP5Z1* versus matched controls. Whole cell lysates were analysed by label-free quantification mass spectrometry. In both cell types, over 7,500 proteins were quantified. The x-axis shows the log₂-fold change between AP-5–deficient and control cells; the y-axis shows the −log₁₀ <i>p</i>-value of significance (2-sided <i>t</i> test). The ‘volcano’ lines indicate the significance threshold (FDR 0.12). For HeLa cells, 3 replicates from control cells and 3 replicates each from 2 independent AP5Z1-knockout cell lines (6 total) were compared (<i>n</i> = 3/6). For fibroblasts, 2 cell lines from patients with null mutations in AP5Z1 were compared to 2 matched control cell lines, each in duplicate (i.e., <i>n</i> = 4). Lysosomal proteins are indicated (salmon-coloured dots); their abundance does not change significantly in AP-5–deficient cells. Data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.s004" target="_blank">S2 Data</a> (HeLa intensity data and Fibroblast intensity data); the plots were generated using Perseus software. (C) Analysis of protein movement on dynamic organellar maps. Control HeLa cells and the 2 independent AP5Z1 knockout lines were subjected to proteomics-based organellar mapping, each in triplicate (see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.s001" target="_blank">S1 Fig</a>). Maps from control and AP5Z1 knockout cells were then compared to detect proteins undergoing shifts in subcellular localization, in which the reproducibility score is a measure of the correlation between replicates of the same clone and between the 2 different AP5Z1 knockout clones. For each protein, the M and R of movement were calculated. Significantly shifting proteins have high M and R scores and are located in the top right quadrant of the MR plot; they are highly enriched in endosomal proteins. The estimated FDR was <0.23 at the cutoffs indicated by the vertical (M score threshold) and horizontal (R score threshold) lines. Subunits of the HOPS (red) and retromer (blue) complexes are highlighted. The cation-independent mannose 6-phosphate receptor (IGFR2R) is a marginal hit (significant at FDR 0.25). The figure focuses on the area of the MR plot in which significant changes are located (R score > 0); see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004411#pbio.2004411.s003" target="_blank">S1 Data</a> (MR plot data) for a complete list of all 2,046 profiled proteins. AP, adaptor protein; CHC, clathrin heavy chain; KO, knockout; M, magnitude; R, reproducibility.</p