Auto-regulation of Secretory Flux by Sensing and Responding to the Folded Cargo Protein Load in the Endoplasmic Reticulum.

Maintaining the optimal performance of cell processes and organelles is the task of auto-regulatory systems. Here we describe an auto-regulatory device that helps to maintain homeostasis of the endoplasmic reticulum (ER) by adjusting the secretory flux to the cargo load. The cargo-recruiting subunit of the coatomer protein II (COPII) coat, Sec24, doubles as a sensor of folded cargo and, upon cargo binding, acts as a guanine nucleotide exchange factor to activate the signaling protein Gα12 at the ER exit sites (ERESs). This step, in turn, activates a complex signaling network that activates and coordinates the ER export machinery and attenuates proteins synthesis, thus preventing large fluctuations of folded and potentially active cargo that could be harmful to the cell or the organism. We call this mechanism AREX (autoregulation of ER export) and expect that its identification will aid our understanding of human physiology and diseases that develop from secretory dysfunction.

PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane.

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11-positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245-dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97- vs. Golgin-245-dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin-mediated cell polarity and cell-cell junctions.

Calcium flow at ER-TGN contact sites facilitates secretory cargo export.

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains mysterious, as the cytosol has a nanomolar steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), allowing protein-mediated exchange of molecular species such as lipids. Here, we show that the TGN export of secretory proteins requires the integrity of ER-TGN MCS and inositol 3 phosphate receptor (IP3R)-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real time. These data show that ER-TGN MCS facilitates the Ca2+ transfer required for Ca2+-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology.

Endomembrane-Based Signaling by GPCRs and G-Proteins.

G-protein-coupled receptors (GPCRs) and G-proteins have a range of roles in many physiological and pathological processes and are among the most studied signaling proteins. A plethora of extracellular stimuli can activate the GPCR and can elicit distinct intracellular responses through the activation of specific transduction pathways. For many years, biologists thought that GPCR signaling occurred entirely on the plasma membrane. However, in recent decades, many lines of evidence have proved that the GPCRs and G-proteins may reside on endomembranes and can start or propagate signaling pathways through the organelles that form the secretory route. How these alternative intracellular signaling pathways of the GPCR and G-proteins influence the physiological and pathological function of the endomembranes is still under investigation. Here, we review the general role and classification of GPCRs and G-proteins with a focus on their signaling pathways in the membrane transport apparatus.

Endogenous and Exogenous Regulatory Signaling in the Secretory Pathway: Role of Golgi Signaling Molecules in Cancer.

The biosynthetic transport route that constitutes the secretory pathway plays a fundamental role in the cell, providing to the synthesis and transport of around one third of human proteins and most lipids. Signaling molecules within autoregulatory circuits on the intracellular membranes of the secretory pathway regulate these processes, especially at the level of the Golgi complex. Indeed, cancer cells can hijack several of these signaling molecules, and therefore also the underlying regulated processes, to bolster their growth or gain more aggressive phenotypes. Here, we review the most important autoregulatory circuits acting on the Golgi, emphasizing the role of specific signaling molecules in cancer. In fact, we propose to draw awareness to highlight the Golgi-localized regulatory systems as potential targets in cancer therapy.

Regulation of cargo export and sorting at the trans-Golgi network.

The sorting and distribution to different final destinations of roughly a third of the membrane and secreted proteins occurs at the level of the trans-Golgi network (TGN). This TGN mission involves efficient mechanisms of cargo recognition and activation of specific signalling pathways. This is important because protein localization is strictly connected with function, and many aberrant phenotypes may occur when a protein is missorted to the wrong cellular compartment. In this review, we briefly summarize the principal players known to be involved in TGN functions, highlighting the importance of regulatory signalling pathways and also the pathological outcomes of aberrant sorting and export events from the TGN compartment.

MDM2-mediated degradation of p14ARF: a novel mechanism to control ARF levels in cancer cells.

We here show a new relationship between the human p14ARF oncosuppressor and the MDM2 oncoprotein. MDM2 overexpression in various cancer cell lines causes p14ARF reduction inducing its degradation through the proteasome. The effect does not require the ubiquitin ligase activity of MDM2 and preferentially occurs in the cytoplasm. Interestingly, treatment with inhibitors of the PKC (Protein Kinase C) pathway and use of p14ARF phosphorylation mutants indicate that ARF phosphorylation could play a role in MDM2 mediated ARF degradation reinforcing our previous observations that ARF phosphorylation influences its stability and biological activity. Our study uncovers a new potentially important mechanism through which ARF and MDM2 can counterbalance each other during the tumorigenic process.

A Golgi-based KDELR-dependent signalling pathway controls extracellular matrix degradation.

We recently identified an endomembrane-based signalling cascade that is activated by the KDEL receptor (KDELR) on the Golgi complex. At the Golgi, the KDELR acts as a traffic sensor (presumably via binding to chaperones that leave the ER) and triggers signalling pathways that balance membrane fluxes between ER and Golgi. One such pathway relies on Gq and Src. Here, we examine if KDELR might control other cellular modules through this pathway. Given the central role of Src in extracellular matrix (ECM) degradation, we investigated the impact of the KDELR-Src pathway on the ability of cancer cells to degrade the ECM. We find that activation of the KDELR controls ECM degradation by increasing the number of the degradative structures known as invadopodia. The KDELR induces Src activation at the invadopodia and leads to phosphorylation of the Src substrates cortactin and ASAP1, which are required for basal and KDELR-stimulated ECM degradation. This study furthers our understanding of the regulatory circuitry underlying invadopodia-dependent ECM degradation, a key phase in metastases formation and invasive growth.

Control systems of membrane transport at the interface between the endoplasmic reticulum and the Golgi.

A fundamental property of cellular processes is to maintain homeostasis despite varying internal and external conditions. Within the membrane transport apparatus, variations in membrane fluxes from the endoplasmic reticulum (ER) to the Golgi complex are balanced by opposite fluxes from the Golgi to the ER to maintain homeostasis between the two organelles. Here we describe a molecular device that balances transport fluxes by integrating transduction cascades with the transport machinery. Specifically, ER-to-Golgi transport activates the KDEL receptor at the Golgi, which triggers a cascade that involves Gs and adenylyl cyclase and phosphodiesterase isoforms and then PKA activation and results in the phosphorylation of transport machinery proteins. This induces retrograde traffic to the ER and balances transport fluxes between the ER and Golgi. Moreover, the KDEL receptor activates CREB1 and other transcription factors that upregulate transport-related genes. Thus, a Golgi-based control system maintains transport homeostasis through both signaling and transcriptional networks.