Mutagenesis of the di-leucine motif in the cytoplasmic tail of newcastle disease virus fusion protein modulates the viral fusion ability and pathogenesis.

BACKGROUND: Newcastle disease virus (NDV) is a highly infectious viral disease, which can affect chickens and many other kinds of birds. The main virulence factor of NDV, the fusion (F) protein, is located on the viral envelope and plays a major role in the virus' ability to penetrate cells and cause host cell fusion during infection. Multiple highly conserved tyrosine and di-leucine (LL) motifs in the cytoplasmic tail (CT) of the virus may contribute to F protein functionality in the viral life cycle. METHODS: To examine the contribution of the LL motif in the biosynthesis, transport, and function of the F protein, we constructed and rescued a NDV mutant strain, rSG10*-F/L537A, with an L537A mutation using a reverse genetic system. Subsequently, we compared the differences in the syncytium formation ability, pathogenicity, and replication levels of wild-type rSG10* and the mutated strain. RESULTS: Compared with rSG10*, rSG10*-F/L537A had attenuated syncytial formation and pathogenicity, caused by a viral budding defect. Further studies showed that the LL-motif mutation did not affect the replication, transcription, or translation of the virus genome but affected the expression of the F protein at the cell surface. CONCLUSIONS: We concluded that the LL motif in the NDV F CT affected the regulation of F protein expression at the cell surface, thus modulating the viral fusion ability and pathogenic phenotype.

Nicotianamine synthase 2 localizes to the vesicles of iron-deficient rice roots, and its mutation in the YXXφ or LL motif causes the disruption of vesicle formation or movement in rice.

Graminaceous plants release mugineic acid family phytosiderophores (MAs) to acquire iron from the soil. Here, we show that deoxymugineic acid (DMA) secretion from rice roots fluctuates throughout the day, and that vesicles accumulate in roots before MAs secretion. We developed transgenic rice plants that express rice nicotianamine (NA) synthase (NAS) 2 (OsNAS2) fused to synthetic green fluorescent protein (sGFP) under the control of its own promoter. In root cells, OsNAS2-sGFP fluorescence was observed in a dot-like pattern, moving dynamically within the cell. This suggests that these vesicles are involved in NA and DMA biosynthesis. A tyrosine motif and a di-leucine motif, which have been reported to be involved in cellular transport, are conserved in all identified NAS proteins in plants. OsNAS2 mutated in the tyrosine motif showed NAS activity and was localized to the vesicles; however, these vesicles stuck together and did not move. On the other hand, OsNAS2 mutated in the di-leucine motif lost NAS activity and did not localize to these vesicles. The amounts of NA and DMA produced and the amount of DMA secreted by OsNAS2-sGFP plants were significantly higher than in non-transformants and domain-mutated lines, suggesting that OsNAS2-sGFP, but not the mutated forms, was functional in vivo. Overall, the localization of NAS to vesicles and the transport of these vesicles are crucial steps in NA synthesis, leading to DMA synthesis and secretion in rice.

A C-terminal di-leucine motif controls plasma membrane expression of PMCA4b.

Recent evidences show that the localization of different plasma membrane Ca(2+) ATPases (PMCAs) is regulated in various complex, cell type-specific ways. Here we show that in low-density epithelial and endothelial cells PMCA4b localized mostly in intracellular compartments and its plasma membrane localization was enhanced upon increasing density of cells. In good correlation with the enhanced plasma membrane localization a significantly more efficient Ca(2+) clearance was observed in confluent versus non-confluent HeLa cell cultures expressing mCherry-PMCA4b. We analyzed the subcellular localization and function of various C-terminally truncated PMCA4b variants and found that a truncated mutant PMCA4b-ct24 was mostly intracellular while another mutant, PMCA4b-ct48, localized more to the plasma membrane, indicating that a protein sequence corresponding to amino acid residues 1158-1181 contained a signal responsible for the intracellular retention of PMCA4b in non-confluent cultures. Alteration of three leucines to alanines at positions 1167-1169 resulted in enhanced cell surface expression and an appropriate Ca(2+) transport activity of both wild type and truncated pumps, suggesting that the di-leucine-like motif (1167)LLL was crucial in targeting PMCA4b. Furthermore, upon loss of cell-cell contact by extracellular Ca(2+) removal, the wild-type pump was translocated to the early endosomal compartment. Targeting PMCA4b to early endosomes was diminished by the L(1167-69)A mutation, and the mutant pump accumulated in long tubular cytosolic structures. In summary, we report a di-leucine-like internalization signal at the C-tail of PMCA4b and suggest an internalization-mediated loss of function of the pump upon low degree of cell-cell contact.

Involvement of a di-leucine motif in targeting of ABCC1 to the basolateral plasma membrane of polarized epithelial cells.

Localization of ATP-binding cassette transporter isoform C1 (ABCC1) to the basolateral membrane of polarized cells is crucial for export of a variety of cellular metabolites; however, the mechanism regulating basolateral targeting of the transporter is poorly understood. Here we describe identification of a basolateral targeting signal in the first cytoplasmic loop domain (CLD1) of human ABCC1. Comparison of the CLD1 amino acid sequences from ABCC1 to ABCC2 revealed that ABCC1 possesses a characteristic sequence, E(295)EVEALI(301), which is comprised of a cluster of acidic glutamate residues followed by a di-leucine motif. This characteristic sequence is highly conserved among vertebrate ABCC1 orthologs and is positioned at a site that is structurally equivalent to the apical targeting signal previously described in ABCC2. Alanine scanning mutagenesis of this sequence in full-length human ABCC1 showed that both L(300) and I(301) residues were required for basolateral targeting of ABCC1 in polarized HepG2 and MDCK cells. Conversely, E(295), E(296), and E(298) residues were not required for basolateral localization of the transporter. Therefore, a di-leucine motif within the CLD1 is a basolateral targeting determinant of ABCC1.

Exposing the Specific Roles of the Invariant Chain Isoforms in Shaping the MHC Class II Peptidome.

The peptide repertoire (peptidome) associated with MHC class II molecules (MHCIIs) is influenced by the polymorphic nature of the peptide binding groove but also by cell-intrinsic factors. The invariant chain (Ii) chaperones MHCIIs, affecting their folding and trafficking. Recent discoveries relating to Ii functions have provided insights as to how it edits the MHCII peptidome. In humans, the Ii gene encodes four different isoforms for which structure-function analyses have highlighted common properties but also some non-redundant roles. Another layer of complexity arises from the fact that Ii heterotrimerizes, a characteristic that has the potential to affect the maturation of associated MHCIIs in many different ways, depending on the isoform combinations. Here, we emphasize the peptide editing properties of Ii and discuss the impact of the various isoforms on the MHCII peptidome.