The Role of Junctophilin Proteins in Cellular Function

Junctophilins (JPHs) comprise a family of structural proteins that connect the plasma membrane to intracellular organelles such as the endo/sarcoplasmic reticulum. Tethering of these membrane structures results in the formation of highly organized subcellular junctions that play important signaling roles in all excitable cell types. There are four JPH isoforms, expressed primarily in muscle and neuronal cell types. Each JPH protein consists of 6 'membrane occupation and recognition nexus' (MORN) motifs, a joining region connecting these to another set of 2 MORN motifs, a putative alpha-helical region, a divergent region exhibiting low homology between JPH isoforms, and a carboxy-terminal transmembrane region anchoring into the ER/SR membrane. JPH isoforms play essential roles in developing and maintaining subcellular membrane junctions. Conversely, inherited mutations in JPH2 cause hypertrophic or dilated cardiomyopathy, while trinucleotide expansions in the JPH3 gene cause Huntington Disease-Like 2. Loss of JPH1 protein levels can cause skeletal myopathy, while loss of cardiac JPH2 levels causes heart failure and atrial fibrillation, among other disease. This review will provide a comprehensive overview of the JPH gene family, phylogeny, and evolutionary analysis of JPH genes and other MORN domain proteins. JPH biogenesis, membrane tethering, and binding partners will be discussed, as well as functional roles of JPH isoforms in excitable cells. Finally, potential roles of JPH isoform deficits in human disease pathogenesis will be reviewed.

Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

Gonadotropin-releasing hormone (GnRH) is the primary neuropeptide controlling reproduction in vertebrates. GnRH stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis via a G protein-coupled receptor, GnRHR, in the pituitary gland. In mammals, GnRHR lacks a C-terminal cytosolic tail (Ctail) and does not exhibit homologous desensitization. This might be an evolutionary adaptation that enables LH surge generation and ovulation. To test this idea, we fused the chicken GnRHR Ctail to the endogenous murine GnRHR in a transgenic model. The LH surge was blunted, but not blocked in these mice. In contrast, they showed reductions in FSH production, ovarian follicle development, and fertility. Addition of the Ctail altered the nature of agonist-induced calcium signaling required for normal FSH production. The loss of the GnRHR Ctail during mammalian evolution is unlikely to have conferred a selective advantage by enabling the LH surge. The adaptive significance of this specialization remains to be determined.

Identification of New In Vivo TonB-FepA Rendezvous Sites

The TonB system of Gram-negative bacteria uses the protonmotive force of the cytoplasmic membrane to energize active transport of large or scarce nutrients across the outer membrane by means of customized beta-barrels known as TonB-dependent transporters (TBDTs). The lumen of each TBDT is occluded by an amino-terminal domain, called the cork, which must be displaced for transport of nutrients or translocation of the large protein toxins that parasitize the system. A complex of cytoplasmic membrane proteins consisting of TonB, ExbB and ExbD harnesses the protonmotive force that TonB transmits to the TBDT. The specifics of this energy transformation are a source of continuing interest. The amino terminal domain of a TBDT contains a region called the TonB box, that is essential for the reception of energy from TonB. This domain is the only identified site of in vivo interaction between the TBDT and TonB, occurring through a non-essential region centered on TonB residue Q160. Because TonB binds to TBDTs whether or not it is active or even intact, the mechanism and extent of cork movement in vivo has been challenging to discover. In this study, we used in vivo disulfide crosslinking between eight engineered Cys residues in Escherichia coli TonB and 42 Cys substitutions in the TBDT FepA, including the TonB box, to identify novel sites of interaction in vivo. The TonB Cys substitutions in the core of an essential carboxy terminal amphipathic helix (residues 199-216) were compared to TonB Q160C interactions. Functionality of the in vivo interactions was established when the presence of the inactive TonB H20A mutation inhibited them. A previously unknown functional interaction between the hydrophilic face of the amphipathic helix and the FepA TonB box was identified. Interaction of Q160C with the FepA TonB box appeared to be less functionally important. The two different parts of TonB also differed in their interactions with the FepA cork and barrel turns. While the TonB amphipathic helix Cys residues interacted only with Cys residues on the periplasmic face of the FepA cork, TonB Q160C interacted with buried Cys substitutions within the FepA cork, the first such interactions seen with any TBDT. Both sets of interactions required active TonB. Taken together, these data suggest a model where the amphipathic helix binds to the TonB box, causing the mechanically weak domain of the FepA cork to dip sufficiently into the periplasmic space for interaction with the TonB Q160 region, which is an interaction that does not occur if the TonB box is deleted. The TonB amphipathic helix also interacted with periplasmic turns between FepA β-strands in vivo supporting a surveillance mechanism where TonB searched for TBDTs on the periplasmic face of the outer membrane.

Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na+/qHCO3− Cotransporter NBCe1

In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+–2HCO3− stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO3− influx. However, NBCe1 in the kidney mediates HCO3− efflux (HCO3− reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3− efflux) than in murine colons (in which NBCe1 mediates HCO3− influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.

A widespread viral entry mechanism: The C-end Rule motif–neuropilin receptor interaction

Many phylogenetically distant animal viruses, including the new coronavirus severe acute respiratory syndrome coronavirus 2, have surface proteins with polybasic sites that are cleaved by host furin and furin-like proteases. Other than priming certain viral surface proteins for fusion, cleavage generates a carboxy-terminal RXXR sequence. This C-end Rule (CendR) motif is known to bind to neuropilin (NRP) receptors on the cell surface. NRPs are ubiquitously expressed, pleiotropic cell surface receptors with important roles in growth factor signaling, vascular biology, and neurobiology, as well as immune homeostasis and activation. The CendR–NRP receptor interaction promotes endocytic internalization and tissue spreading of different cargo, including viral particles. We propose that the interaction between viral surface proteins and NRPs plays an underappreciated and prevalent role in the transmission and pathogenesis of diverse viruses and represents a promising broad-spectrum antiviral target.

Trypanosome morphogenesis involves recruitment of a pleckstrin homology domain protein by an orphan kinesin to the microtubule quartet

ABSTRACTKinesins are motor proteins found in all eukaryotic lineages that move along microtubule tracks to mediate numerous cellular processes such as mitosis and intracellular transport of cargo. In trypanosomatids, the kinesin protein superfamily has undergone a prominent expansion, giving these protists one of the most diverse kinesin repertoires. This has led to the emergence of two trypanosomatid-restricted groups of kinesins. Here, we characterize in Trypanosoma brucei TbKifX2, a hitherto orphaned kinesin that belongs to one of these groups. Representing a rare instance, TbKifX2 tightly interacts with TbPH1, a kinesin-like protein with an inactive motor domain. TbPH1 is named after a pleckstrin homology (PH) domain present within its carboxy-terminal tail. TbKifX2 recruits TbPH1 to the microtubule quartet (MtQ), a characteristic but poorly understood cytoskeletal structure that is part of the multipartite flagellum attachment zone (FAZ) and extends from the basal body to the anterior of the cell body. The proximal proteome of TbPH1 is comprised of four proteins that localize to the FAZ, consistent with the notion that the TbKifX2/TbPH1 complex are the first identified proteins to bind the MtQ along its whole length. Simultaneous ablation of both TbKifX2 and TbPH1 leads to the formation of prominent protrusions from the cell posterior. Thus, these two trypanosomatid-restricted proteins, which specifically localize to the MtQ in a microtubule-rich cell, appear to be contributors to morphogenesis in T. brucei.IMPORTANCETrypanosomatids are a group of unicellular parasites that infect a wide range of hosts from land plants to animals. They are also eukaryotes that have been shaped by prolonged independent evolution since this domain of life has radiated from a common ancestor almost 2 billion years ago. Thus, any resulting unique biological properties can be potentially exploited for treatment of infectious diseases caused by trypanosomatids. The cytoskeleton of trypanosomatids represents an ancient organelle that has undergone such modification. Here, we show that two trypanosomatid-specific proteins named TbPH1 and TbKifX2 form a complex that localizes to the microtubule quartet, a cytoskeletal structure characteristic to trypanosomatids. Ablation of these proteins in Trypansoma brucei leads to distinct morphological defects, making them not only intrinsically interesting topics of study, but potential therapeutic targets as well.

Characterization of nuclear pore complex targeting domains in Pom152 in Saccharomyces cerevisiae

Pom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in pom152Δ cells results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.

Addition of a carboxy terminal tail to the normally tailless gonadotropin-releasing hormone receptor impairs fertility in female mice

Gonadotropin-releasing hormone (GnRH) is the primary neuropeptide controlling reproduction in vertebrates. GnRH stimulates follicle-stimulating hormone (FSH) and luteinizing hormone (LH) synthesis via a G protein-coupled receptor, GnRHR, in the pituitary gland. In mammals, GnRHR lacks a C-terminal cytosolic tail (Ctail) and does not exhibit homologous desensitization. This might be an evolutionary adaptation that enables LH surge generation and ovulation. To test this idea, we fused the chicken GnRHR Ctail to the endogenous murine GnRHR in a transgenic model. The LH surge was blunted, but not blocked in these mice. In contrast, they showed reductions in FSH production, ovarian follicle development, and fertility. Addition of the Ctail altered the nature of agonist-induced calcium signaling required for normal FSH production. The loss of the GnRHR Ctail during mammalian evolution is unlikely to have conferred a selective advantage by enabling the LH surge. The adaptive significance of this specialization remains to be determined.