LDL receptor related protein 1 requires the I3 domain of discs-large homolog 1/DLG1 for interaction with the kinesin motor protein KIF13B.

KIF13B, a kinesin-3 family motor, was originally identified as GAKIN due to its biochemical interaction with human homolog of Drosophila discs-large tumor suppressor (hDLG1). Unlike its homolog KIF13A, KIF13B contains a carboxyl-terminal CAP-Gly domain. To investigate the function of the CAP-Gly domain, we developed a mouse model that expresses a truncated form of KIF13B protein lacking its CAP-Gly domain (KIF13BΔCG), whereas a second mouse model lacks the full-length KIF13A. Here we show that the KIF13BΔCG mice exhibit relatively higher serum cholesterol consistent with the reduced uptake of [3H]CO-LDL in KIF13BΔCG mouse embryo fibroblasts. The plasma level of factor VIII was not significantly elevated in the KIF13BΔCG mice, suggesting that the CAP-Gly domain region of KIF13B selectively regulates LRP1-mediated lipoprotein endocytosis. No elevation of either serum cholesterol or plasma factor VIII was observed in the full length KIF13A null mouse model. The deletion of the CAP-Gly domain region caused subcellular mislocalization of truncated KIF13B concomitant with the mislocalization of LRP1. Mechanistically, the cytoplasmic domain of LRP1 interacts specifically with the alternatively spliced I3 domain of DLG1, which complexes with KIF13B via their GUK-MBS domains, respectively. Importantly, double mutant mice generated by crossing KIF13A null and KIF13BΔCG mice suffer from perinatal lethality showing potential craniofacial defects. Together, this study provides first evidence that the carboxyl-terminal region of KIF13B containing the CAP-Gly domain is important for the LRP1-DLG1-KIF13B complex formation with implications in the regulation of metabolism, cell polarity, and development.

RsgA couples the maturation state of the 30S ribosomal decoding center to activation of its GTPase pocket.

During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgA΄s mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates. Moreover, RsgA exploits its distinct GTPase pocket and specific interactions with the 30S to coordinate GTPase activation with the maturation state of the 30S subunit. This coordination validates the architecture of the decoding center and facilitates the timely release of RsgA to control the progression of 30S biogenesis.

Complete Genome Sequence of the Porcine Epidemic Diarrhea Virus Variant Tottori2/JPN/2014.

Porcine epidemic diarrhea virus (PEDV) is a cause of diarrhea outbreaks at swine farms, causing vomiting, severe diarrhea, and mortality in piglets. We sequenced and analyzed the complete genome of recently isolated strains. Tottori2/JPN/2014, one of the sequenced PEDV strains, had a unique large deletion in the S gene.

Impairment of ribosome maturation or function confers salt resistance on Escherichia coli cells.

We found that loss of integrity of the ribosome by removal of a putative ribosome maturation factor or a ribosomal protein conferred salt tolerance on Escherichia coli cells. Some protein synthesis inhibitors including kasugamycin and chloramphenicol also had a similar effect, although kasugamycin affected neither 16S rRNA maturation nor subunit association into a 70S ribosome. Thus, salt tolerance is a common feature of cells in which maturation or function of the ribosome is impaired. In these cells, premature induction of an alternative sigma factor, σ(E), by salt stress was observed. These results suggest the existence of a yet-unknown stress response pathway mediated by the bacterial ribosome.

Physiological role of RsgA in ribosome biosynthesis.

RsgA is a unique GTP hydrolytic protein, in which the GTPase activity is significantly enhanced by the small ribosomal subunit. Depletion of RsgA causes slow cell growth as well as defects in the subunit assembly of the ribosome and the 16S rRNA processing, suggesting its involvement in the maturation of the small subunit. Several antibiotics bound to the decoding center of the small subunit inhibited the ribosome-dependent GTPase activity of RsgA. Our recent study using chemical modification indicates that the binding of RsgA induces conformational changes around the A site, P site, and helix 44. These results suggest that RsgA is involved in the maturation step of the decoding center of the small subunit of ribosome. Here, we also show a physiological role of RsgA under stress condition.

Removal of a ribosome small subunit-dependent GTPase confers salt resistance on Escherichia coli cells.

RsgA is a unique GTP hydrolytic protein in which GTPase activity is significantly enhanced by the small ribosomal subunit. Deletion of RsgA causes slow cell growth as well as defects in subunit assembly of the ribosome and 16S rRNA processing, suggesting its involvement in maturation of the small subunit. In this study, we found that removal of RsgA or inactivation of its ribosome small subunit-dependent GTPase activity provides Escherichia coli cells with resistance to high salt stress. Salt stress suppressed the defects in subunit assembly of the ribosome and processing of 16S rRNA as well as truncation of the 3' end of 16S rRNA in RsgA-deletion cells. In contrast, salt stress transiently impaired subunit assembly of the ribosome and processing of 16S rRNA and induced 3' truncation of 16S rRNA in wild-type cells. These results suggest that the action of RsgA on the ribosome, which usually facilitates maturation of the small subunit, disturbs it under a salt stress condition. Consistently, there was a drastic but transient decrease in the intracellular amount of RsgA after salt shock. Salt shock would make the pathway of maturation of the ribosome small subunit RsgA independent.

Ribosome-small-subunit-dependent GTPase interacts with tRNA-binding sites on the ribosome.

RsgA (ribosome-small-subunit-dependent GTPase A, also known as YjeQ) is a unique GTPase in that guanosine triphosphate hydrolytic activity is activated by the small subunit of the ribosome. Disruption of the gene for RsgA from the genome affects the growth of cells, the subunit association of the ribosome, and the maturation of 16S rRNA. To study the interaction of Escherichia coli RsgA with the ribosome, chemical modifications using dimethylsulfate and kethoxal were performed on the small subunit in the presence or in the absence of RsgA. The chemical reactivities at G530, A790, G925, G926, G966, C1054, G1339, G1405, A1413, and A1493 in 16S rRNA were reduced, while those at A532, A923, G1392, A1408, A1468, and A1483 were enhanced, by the addition of RsgA, together with 5'-guanylylimidodiphosphate. Among them, the chemical reactivities at A532, A790, A923, G925, G926, C1054, G1392, A1413, A1468, A1483, and A1493 were not changed when RsgA was added together with GDP. These results indicate that the binding of RsgA induces conformational changes around the A site, P site, and helix 44, and that guanosine triphosphate hydrolysis induces partial conformational restoration, especially in the head, to dissociate RsgA from the small subunit. RsgA has the capacity to coexist with mRNA in the ribosome while it promotes dissociation of tRNA from the ribosome.