GPSM2-GNAI Specifies the Tallest Stereocilia and Defines Hair Bundle Row Identity Highlights d GPSM2, GNAI, WHRN, MYO15A, and EPS8 work in the same pathway to shape hair bundles d GPSM2-GNAI-WHRN is a late module added to MYO15A-EPS8 at row 1 stereocilia tips only d GPSM2-GNAI defines the identity of the tallest, first-row stereocilia d Mutant bundles comprise generic stereocilia lacking differential row identity

Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism that regulates a plethora of physiological processes. Recent studies unveiled an unconventional type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases in the canonical ubiquitination pathway, here we show that 2 paralogous Legionella effectors, Lpg2154 (DupA; deubiquitinase for PR-ubiquitination) and Lpg2509 (DupB), reverse PR-ubiquitination by specific removal of phosphoribosyl-Ub from substrates. Both DupA and DupB are fully capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA in mammalian cells. We further show that deletion of these 2 genes results in significant accumulation of PR-ubiquitinated species in host cells infected with Legionella. In addition, we have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play a role in modulating the association of PR-ubiquitinated host targets with Legionella-containing vacuoles. Together, our data establish a complete PR-ubiquitination and deubiquitination cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent posttranslational modification.

Summary Ubiquitination is a post-translational modification that regulates a myriad of cellular processes in eukaryotes1–4. The conventional ubiquitination cascade culminates in a covalent linkage between the C-terminus of ubiquitin (Ub) and a target protein, most often on a lysine sidechain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a novel mode of ubiquitination in which a phosphoribosylated ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6–8. To uncover the molecular mechanism of this unique post-translational modification, we determined the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain (PDE) and a mono-ADP-ribosyltransferase (mART) domain. Biochemical analysis shows that the mART domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the PDE domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous PDE domain from the SidE family member SdeD9 in complex with Ub or ADPR-Ub. The structures suggest an intriguing mechanism for how SdeA processes ADPR-Ub to PR-Ub plus AMP and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-ubiquitination (PR-ubiquitination) and paves the way for future studies of this unusual type of ubiquitination in eukaryotes.

Hair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.

24Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism 25 that regulates a plethora of physiological processes. Recent studies unveiled an unconventional 26 type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as 27 SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a 28 phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases 29 (DUBs) in the canonical ubiquitination pathway, here we show that two Legionella effectors, 30 named DupA (deubiquitinase for PR-ubiquitination) and DupB, reverse PR-ubiquitination by 31 specific removal of phosphoribosyl-Ub (PR-Ub) from substrates. Both DupA and DupB are fully 32 capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA 33 in mammalian cells. We further show that deletion of these two genes results in significant 34 accumulation of PR-ubiquitinated species in host cells infected with Legionella. In addition, we 35 have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play 36 a role in modulating the association of PR-ubiquitinated host targets with Legionella containing 37 vacuoles (LCV). Together, our data establish a complete PR-ubiquitination and deubiquitination 38 cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent 39 posttranslational modification.40 3 Statement of significance 41 Ubiquitination is a vital posttranslational modification in eukaryotes. A variety of 42 microbial pathogens exploit this pathway during their infection. Legionella pneumophila, the 43 causative bacterial pathogen of Legionnaires' disease, has been show to hijack host ubiquitination 44 pathway via a large number of effectors. Recent studies revealed a family of effectors catalyzing 45 a novel type of Ub-dependent posttranslational modification, namely PR-ubiquitination. Here we 46 report two new players, DupA and DupB, involved in this unconventional pathway. We found that 47 DupA and DupB function as PR-Ub specific DUBs and play a role in regulating the PR-48 ubiquitination levels of host targets. Our results not only provide an expanding view of the PR-49 ubiquitination pathway, but may also facilitate the future identification of PR-ubiquitination 50 pathways in eukaryotes. 51 4 \body 52 Introduction 53Ubiquitin (Ub), a 76 amino acid protein, is attached to specific proteins as a potent 54 posttranslational mechanism. Ubiquitination plays an essential role in a broad aspect of cellular 55 processes, including protein homeostasis [1], cell signaling [2], and membrane trafficking [3, 4]. 56 Following the conventional scheme of ubiquitination, Ub is covalently coupled to lysine residues 57 on target proteins via the sequential activities of a collection of enzymes known as E1, E2, and 58 E3s [5]. The C-terminal glycine residue of Ub is first activated and covalently linked to the 59 catalytic cysteine residue of the Ub act...

Inhibitory G proteins (GNAI/Gα i ) bind to the scaffold G protein signaling modulator 2 (GPSM2) to form a conserved polarity complex that regulates cytoskeleton organization. GPSM2 keeps GNAI in a guanosine diphosphate (GDP)-bound state, but how GPSM2-GNAI is generated or relates to heterotrimeric G protein signaling remains unclear. We find that RGS12, a GTPase-activating protein (GAP), is required to polarize GPSM2-GNAI at the hair cell apical membrane and to organize mechanosensory stereocilia in rows of graded heights. Accordingly, RGS12 and the guanine nucleotide exchange factor (GEF) DAPLE are asymmetrically co-enriched at the hair cell apical junction, and Rgs12 mouse mutants are deaf. GPSM2 and RGS12 share GoLoco motifs that stabilize GNAI(GDP), and GPSM2 outcompetes RGS12 to bind GNAI. Our results suggest that polarized GEF/GAP junctional activity might dissociate heterotrimeric G proteins, generating free GNAI(GDP) for GPSM2 at the adjacent apical membrane. GPSM2-GNAI(GDP), in turn, imparts asymmetry to the forming stereocilia to enable sensory function in hair cells.

Heterotrimeric G-proteins, consisting of alpha, beta, and gamma subunits, participate in major signaling pathways in eurkaryotes and mammals, including those in the nervous and sensory systems. G proteins are activated upon stimulation of G-protein coupled receptors and function as molecular switches through nucleotide cycling by the alpha subunit and by their interactions with intracellular effectors. The mechanism of activation and deactivation of the mammalian heterotrimer is well understood through extensive structural and functional studies, and is being used as a basis for predictions for mechanisms involving the plant heterotrimer. Recent evidence, however, indicates significant differences in the two systems [1]. We undertook a structural investigation of the A. thaliana complex in order to gain insights for G-protein activation and the molecular interactions in the G-protein related signaling pathways in plants. The alpha subunit GPA1 was cloned and expressed in P. pastoris and purified with high yield and homogeneity in functional form. Similarly the beta and gamma subunits (AGB1 and AGG2) were cloned and expressed in E. Coli. AGB1 expression resulted in production of unfolded protein whereas AGG2 could be obtained on its own with high yield and purity. Biochemical analyses and biophysical studies using SAXS, CD, DLS, UV-vis spectroscopy reveal structural parameters of GPA1 for comparison with its mammalian counterparts and indicate the propensity of GPA1 to form oligomers in solution. After purification form E. coli AGG2, can be stabilized in dimeric form and has a flexible and extended structure. Structure of the dimer is sensitive to the concentration of salt and reducing agent in the solution.

The double helix of DNA or RNA when composed of purinepyrimidine repeats, can adopt a left-handed helical structure called Z-DNA and Z-RNA respectively [1]. Such dinucleotide repeats in genomic sequences have been associated with instability leading to cancer, for reasons not entirely understood. Adoption of the left handed conformation in just a region of a polynucleotide sequence results in the formation of conformational junctions: a B-Z junction is formed at the boundaries of the left handed helix [2], a Z-Z junction is commonly formed in sequences where the dinucleotide repeat is interrupted by single base insertions or deletions that bring neighboring left-handed helices out of phase. We report the first crystal structure of a Z-Z junction stabilized by Zα, the Z-DNA binding domain of the RNA editing enzyme ADAR1. The junction structure consists of a single base-pair and leads to partial or full disruption of the helical stacking. The junction region allows intercalating agents to insert themselves in the otherwise resistant to intercalation left-handed helix. However, unlike a B-Z junction the bases do not become fully extruded and the stacking between the two left handed helices is not continuous [2].