J D Lee

PHAGOCYTOSIS OF GRAM-NEGATIVE BACTERIA BY A UNIQUE CD14-DEPENDENT MECHANISM

THP‐l‐derived cell lines were stably transfected with constructs encoding glycophosphatidylinositol (GPI) ‐anchored or transmembrane forms of human CD14. CD14 expression was associated with enhanced phagocytosis of serum (heat‐inactivated) ‐opsonized Escherichia coli(opEc). Both the CPI‐anchored and transmembrane forms of CD14 supported phagocytosis of opEc equally well. Lipopolysaccharide‐binding protein (LBP) played a role in CD14‐dependent phagocytosis as evidenced by inhibition of CD14‐dependent phagocytosis of opEc with anti‐LBP monoclonal antibody (mAb) and by enhanced phagocytosis of E. coliopsonized with purified LBP. CD14‐dependent phagocytosis was inhibited by a phosphatidylinositol (PI) 3‐kinase inhibitor (wortmannin) and a protein tyrosine kinase inhibitor (tyrphostin 23) but not a protein kinase C inhibitor (bisindolylmaleimide) or a divalent cation chelator (ethylenediaminetetraacetate). Anti‐LBP mAb 18G4 and anti‐CD 14 mAb 18E12 were used to differentiate between the pathways involved in CD14‐dependent phagocytosis and CD14‐dependent cell activation. F(ab′)2 fragments of 18G4, a mAb to LBP that does not block cell activation, inhibited ingestion of opEc by THP1‐wtCD14 cells. 18E12 (an anti‐CD14 mAb that does not block LPS binding to CD14 but does inhibit CD14‐dependent cell activation) did not inhibit phagocytosis of LBP‐opEc by THP1‐wtCD14 cells. Furthermore, CD14‐dependent phagocytosis was not inhibited by anti‐CD 18 (CR3 and CR4 β‐chain) or anti‐Fcγ receptor mAb. J. Leukoc. Biol. 62: 786–794; 1997.

Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy

The E3 ligase Ltn1 and the deubiquitylase Ubp3-Bre5 titrate the level of ribosomal subunit ubiquitylation and thereby set the rate of ribosomal protein degradation by ribophagy in response to nutrient supply and the level of protein translation.

Single-particle EM reveals extensive conformational variability of the Ltn1 E3 ligase

Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its evolutionarily conserved large size, Ltn1 is characterized by the presence of a conserved N terminus, HEAT/ARM repeats predicted to comprise the majority of the protein, and a C-terminal catalytic RING domain, although the protein’s exact structure is unknown. We used numerous single-particle EM strategies to characterize Ltn1’s structure based on negative stain and vitreous ice data. Two-dimensional classifications and subsequent 3D reconstructions of electron density maps show that Ltn1 has an elongated form and presents a continuum of conformational states about two flexible hinge regions, whereas its overall architecture is reminiscent of multisubunit cullin–RING ubiquitin ligase complexes. We propose a model of Ltn1 function based on its conformational variability and flexibility that describes how these features may play a role in cotranslational protein quality control.

Structure and function of the yeast listerin (Ltn1) conserved N-terminal domain in binding to stalled 60S ribosomal subunits

Significance The listerin (Ltn1) E3 ubiquitin ligase ubiquitylates and promotes degradation of aberrant nascent chains that become stalled on ribosomal 60S subunits. Ltn1-dependent nascent chain ubiquitylation was reconstituted in vitro using extracts of genetically manipulated Neurospora strains. Such extracts, supplemented or not with recombinant factors (such as Ltn1 from Saccharomyces cerevisiae), represent a new system to study ribosome-associated protein quality control. Utilizing this system, we show that mutations in Ltn1’s conserved N-terminal domain result in defective 60S binding and nascent chain ubiquitylation, without affecting Ltn1’s intrinsic E3 activity. Furthermore, we have solved the crystal structure of Ltn1’s N-terminal domain, which provides detailed information and insights into how Ltn1 interacts with stalled 60S subunits. Our observations shed light on how cells handle protein quality control substrates. The Ltn1 E3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. Ltn1 binds to 60S ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among Ltn1’s substrates are aberrant products of mRNA lacking stop codons [nonstop translation products (NSPs)]. Here, we report the reconstitution of NSP ubiquitylation in Neurospora crassa cell extracts. Upon translation in vitro, ribosome-stalled NSPs were ubiquitylated in an Ltn1-dependent manner, while still ribosome-associated. Furthermore, we provide biochemical evidence that the conserved N-terminal domain (NTD) plays a significant role in the binding of Ltn1 to 60S ribosomal subunits and that NTD mutations causing defective 60S binding also lead to defective NSP ubiquitylation, without affecting Ltn1’s intrinsic E3 ligase activity. Finally, we report the crystal structure of the Ltn1 NTD at 2.4-Å resolution. The structure, combined with additional mutational studies, provides insight to NTD’s role in binding stalled 60S subunits. Our findings show that Neurospora extracts can be used as a tool to dissect mechanisms underlying ribosome-associated protein quality control and are consistent with a model in which Ltn1 uses 60S subunits as adapters, at least in part via its NTD, to target stalled NSPs for ubiquitylation.