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.

Adenosine diphosphate (ADP) ribosylation is a protein posttranslational modification (PTM) consisting of the transfer of an ADP-ribose moiety from NAD⁺ to target amino acids that is highly conserved throughout evolution (1–3). The enzymes catalyzing this reaction, named ADP-ribosyltransferases (ARTs), first diversified in bacteria into a variety of systems involved in defensive and offensive strategies in intragenomic, intergenomic, and intraorganismal conflicts, and have been acquired by eukaryotes from these conflict systems several times throughout evolution (1, 4). In eukaryotes, ADP-ribosyltransferases are often components of core regulatory and epigenetic processes (5–7). The analysis of their eukaryotic substrates is thus likely to provide information on the organization and regulation of key cellular functions.The ARTs (8) constitute a major family of ADP-ribosyltransferases whose members catalyze ADP-ribosylation by adding either single or multiple units of the NAD⁺-deriving ADP-ribose onto target proteins respectively, mono- and poly-ADP-ribosylation, hereafter referred to as MARylation and PARylation (9)]. MARylation of mammalian proteins was first discovered decades ago to mediate the pathogenic action of bacterial toxins in host cells (10, 11). The endogenous occurrence of this PTM in mammalian cells later became evident (11–16) and, recently, with the definition of the different enzymes catalyzing the reaction, the cellular functions it regulates are emerging (17–19).So far, eukaryotic ADP-ribosylation has been mainly studied under stress conditions, as exemplified by the role of poly (ADP-ribose) polymerase 1 (PARP1)–mediated PARylation during the DNA-damage response (20), PARP5, -12, and -13 in stress-granule formation (21–23), or PARP16 in the unfolded protein response (24, 25), while its impact on physiological cellular processes remains poorly defined.Intracellular membrane transport is emerging as a function regulated by PARPs, with particular reference to Golgi-localized PARPs, namely PARP5 and -12 (26). PARP5 (also called tankyrase) is known to regulate the delivery of the glucose transporter GLUT4 from the trans-Golgi network (TGN) to glucose-storage vesicles and thus to the plasma membrane PM (27–30)]. PARP12, originally described to be involved in defense against viral infections (31–34), is involved in the anterograde transport of the vesicular stomatitis virus G protein (VSVG) from the TGN to the PM (21, 26, 35) and is a well-known component of stress granules, where it translocates from the Golgi upon oxidative stress (21, 23).The TGN is a major sorting station where cargoes are conveyed and sorted into distinct transport carriers for trafficking to post-Golgi compartments and to the PM (36). The different trafficking routes undertaken by individual cargoes are regulated by transport machineries, including small G proteins belonging to the ADP-ribosylation factor (Arf) and Rab families, cytosolic cargo-adaptor proteins, coat proteins, and accessory proteins, all involved in cargo “packaging” into specific transport carriers to achieve correct sorting and delivery (36–38).Here, we report that PARP12 controls the basolateral transport of a subclass of basolateral cargoes, which includes VSVG and E-cadherin, through the MARylation of Golgin-97. Moreover, we find that PARP12-mediated MARylation requires the presence of protein kinase D (PKD), a master regulator of basolateral transport (39, 40), and that it is stimulated by PKD during cargo trafficking. Traffic-activated PKD phosphorylates PARP12, activating its enzymatic activity. It thus emerged that PARP12-mediated MARylation of Golgin-97 is a component of the PKD-dependent regulatory network underlying the basolateral secretion of a select subgroup of cargo proteins, including E-cadherin. Since E-cadherin is required for the formation of proper adherens junctions and epithelial polarization, we propose that the regulatory cascade described in this study may play a role in the maintenance of cellular polarity in epithelial cells and therefore in various body functions (41, 42).