Phosphoregulation accommodates Type III secretion and assembly of a tether of ER-Chlamydia inclusion membrane contact sites

Abstract

Membrane contact sites (MCS) are crucial for nonvesicular trafficking-based interorganelle communication. Endoplasmic reticulum (ER)–organelle tethering occurs in part through the interaction of the ER resident protein VAP with FFAT motif-containing proteins. FFAT motifs are characterized by a seven amino acidic core surrounded by acid tracks. We have previously shown that the human intracellular bacterial pathogen Chlamydia trachomatis establishes MCS between its vacuole (the inclusion) and the ER through expression of a bacterial tether, IncV, displaying molecular mimicry of eukaryotic FFAT motif cores. Here, we show that multiple layers of host cell kinase-mediated phosphorylation events govern the assembly of the IncV–VAP tethering complex and the formation of ER-Inclusion MCS. Via a C-terminal region containing three CK2 phosphorylation motifs, IncV recruits CK2 to the inclusion leading to IncV hyperphosphorylation of the noncanonical FFAT motif core and serine-rich tracts immediately upstream of IncV FFAT motif cores. Phosphorylatable serine tracts, rather than genetically encoded acidic tracts, accommodate Type III-mediated translocation of IncV to the inclusion membrane, while achieving full mimicry of FFAT motifs. Thus, regulatory components and post-translational modifications are integral to MCS biology, and intracellular pathogens such as C. trachomatis have evolved complex molecular mimicry of these eukaryotic features.

Homologues of the Chlamydia trachomatis and Chlamydia muridarum Inclusion Membrane Protein IncS Are Interchangeable for Early Development but Not for Inclusion Stability in the Late Developmental Cycle

“…Through gain- and loss-of-function studies, we showed that the C. trachomatis inclusion membrane protein IncS Ct interacts with, recruits, and colocalizes with STIM1 at the inclusion, thus identifying IncS Ct as a novel component of ER-inclusion MCS. Identification of the IncS Ct -STIM1 complex is in line with the current model that ER-inclusion MCS localized protein complexes are composed of specific inclusion membrane proteins that each interact with a defined set of host factors ( 17 , 24 , 25 ).…”

“…While the respective role of the IncD-CERT-VAP and IncV-VAP complexes in lipid acquisition and ER-inclusion tethering is fairly well established ( 17 , 23 – 25 ), the role of the IncS Ct -STIM1 complex remains elusive. The concomitant expression and inclusion localization of IncS Ct with the recruitment of STIM1 to the inclusion, starting early and lasting throughout the C. trachomatis developmental cycle ( 26 ), are in line with a shared function for IncS Ct and STIM1.…”

“…Another complex consists of the Inc protein IncV and the ER resident host protein, VAP and constitutes a structural component that tethers the ER to the inclusion membrane ( 24 ). The assembly of the IncV-VAP complex is subjected to regulation via posttranslational phosphorylation by the host kinase CK2 ( 25 ).…”

The Chlamydia trachomatis type III-secreted effector protein CteG induces centrosome amplification through interactions with centrin-2

“…Protein purification and pulldowns were conducted as previously described ( 55 ) with minor modifications. Expression of GST-CETN2, MBP, MBP-CT105, or MBP-CT229 was induced overnight by the addition of isopropyl-β-thiogalactopyranoside (1 mM, final concentration) to a 1 L culture E. coli BL21-λDE3 at OD 0.8.…”

Legionella‐ and host‐driven lipid flux at LCV‐ER membrane contact sites promotes vacuole remodeling

“…Intriguingly, the Chlamydia integral membrane proteins IncV and IncD tether the inclusion to the ER (Stanhope & Derre, 2018). IncV directly binds Vap on the ER through a FFAT motif (Stanhope et al , 2017), and the effector recruits the host protein kinase CK2 to the inclusion leading to the hyperphosphorylation of a FFAT motif core and serine‐rich segments immediately upstream of the noncanonical and canonical FFAT motifs, thus promoting the IncV‐Vap interaction (Ende et al , 2022). IncD indirectly makes contact to Vap through the FFAT motif‐containing host protein CERT (ceramide transfer protein; Derre et al , 2011; Elwell et al , 2011).…”