Arabidopsis cyclophilins direct plasmodesmata-targeting of mobile mRNA via organelle hitchhiking

Plants selectively transport mobile mRNAs through intercellular pores, plasmodesmata (PD), to distribute spatial information for synchronizing meristematic differentiation with environmental dynamics. However, how plants recognize and deliver mobile mRNAs to PD remains unknown. Here, by using RNA-live cell imaging, we show that mobile mRNAs hitchhike on organelle trafficking to transport to PD. Perturbed cytoskeleton organization or organelle trafficking severely disrupts the subcellular distribution of mobile mRNAs. We further show that Arabidopsis rotamase cyclophilins (ROCs), which are organelle-localized RNA-binding proteins (RBPs), specifically bind mobile mRNAs on the surface of organelles to direct PD-targeting. Arabidopsis roc quadruple mutants showed reduced in PD-targeting of mobile mRNAs, along with phenotype alterations. ROCs can move intercellularly and form RNA-protein complexes in phloem, suggesting the roles of ROCs in delivery of mobile mRNAs through PD. Our results highlight that an RBP-mediated hitchhiking system is purposely recruited to orient plant-mobile mRNAs to PD for intercellular transport.

Structural and Functional Characterization of Legionella pneumophila Effector MavL

Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose “reader” proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitin-conjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila.

Impaired Mitochondrial Mobility in Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth (CMT) disease is a progressive, peripheral neuropathy and the most commonly inherited neurological disorder. Clinical manifestations of CMT mutations are typically limited to peripheral neurons, the longest cells in the body. Currently, mutations in at least 80 different genes are associated with CMT and new mutations are regularly being discovered. A large portion of the proteins mutated in axonal CMT have documented roles in mitochondrial mobility, suggesting that organelle trafficking defects may be a common underlying disease mechanism. This review will focus on the potential role of altered mitochondrial mobility in the pathogenesis of axonal CMT, highlighting the conceptional challenges and potential experimental and therapeutic opportunities presented by this “impaired mobility” model of the disease.

Intellectual disability-associated gain-of-function mutations in CERT1 that encodes the ceramide transport protein CERT

Intellectual disability (ID) is a developmental disorder that includes both intellectual and adaptive functioning deficits in conceptual, social, and practical domains. Although evidence-based interventions for patients have long been desired, their progress has been hindered due to various determinants. One of these determinants is the complexity of the origins of ID. The ceramide transport protein (CERT) encoded by CERT1 mediates inter-organelle trafficking of ceramide for the synthesis of intracellular sphingomyelin. Utilizing whole exome sequencing analysis, we identified a novel CERT variant, which substitutes a serine at position 135 (S135) for a proline in a patient with severe ID. Biochemical analysis showed that S135 is essential for hyperphosphorylation of a serine-repeat motif of CERT, which is required for down-regulation of CERT activity. Amino acid replacements of S135 abnormally activated CERT and induced an intracellular punctate distribution pattern of this protein. These results identified specific ID-associated CERT1 mutations that induced gain-of-function effects on CERT activity. These findings provide a possible molecular basis for not only new diagnostics but also a conceivable pharmaceutical intervention for ID disorders caused by gain-of-function mutations in CERT1.

The bacterial deubiquitinase Ceg23 regulates the association of Lys-63–linked polyubiquitin molecules on the Legionella phagosome

Legionella pneumophila is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63–linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63–chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 Å resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. ceg23 deletion increased the association of Lys-63–linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63–linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the Legionella phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of L. pneumophila and Ceg23's role in bacterial virulence.