Distribution of microplastics in bathyal- to hadal-depth sediments and transport process along the deep-sea canyon and the Kuroshio Extension in the Northwest Pacific.

Understanding microplastic (MP) behavior in oceans is crucial for reducing marine plastic pollution. However, the complex process underlying MP transportation to the deep seafloor remains unknown despite the deep sea being considered its major sink. We focused on MP distribution in Sagami Bay (adjacent to highly populated areas of Japan), the plate triple junction connected through the Sagami Trough, and the abyssal plain immediately below the Kuroshio Extension. We observed the highest number of MPs in the abyssal stations, more than previously reported. The polymer types and aspect ratio of MPs in the abyssal stations significantly differed from those in the bathyal/hadal stations. The study suggests that MPs accumulated in the open ocean surface layer sink to the abyssal plains immediately below it, while MPs from land sources accumulate in the bathyal depth and are transported to the hadal depth near the coast through turbidity currents along the submarine canyon.

Vertical-scanning mutagenesis of amino acids in a model N-myristoylation motif reveals the major amino-terminal sequence requirements for protein N-myristoylation.

In order to determine the amino-terminal sequence requirements for protein N-myristoylation, site-directed mutagenesis of the N-terminal region was performed using tumor necrosis factor (TNF) mutants as model substrate proteins. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system using rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAAAAA at its N-terminus was used as the starting sequence to identify elements critical for protein N-myristoylation. Sequential vertical-scanning mutagenesis of amino acids at a distinct position in this model N-terminal sequence revealed the major sequence requirements for protein N-myristoylation: the combination of amino acids at position 3 and 6 constitutes a major determinant for the susceptibility to protein N-myristoylation. When Ser was located at position 6, 11 amino acids (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) were permitted at position 3 to direct efficient protein N-myristoylation. In this case, the presence of Lys at position 7 was found to affect the amino acid requirement at position 3 and Lys became permitted at this position. When Ser was not located at position 6, only 3 amino acids (Ala, Asn, Gln) were permitted at position 3 to direct efficient protein N-myristoylation. The amino acid requirements found in this study were fully consistent with the N-terminal sequence of 78 N-myristoylated proteins in which N-myristoylation was experimentally verified. These observations strongly indicate that the combination of amino acids at position 3, 6 and 7 is a major determinant for protein N-myristoylation.

C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and targeted to mitochondria.

To detect the posttranslational N-myristoylation of caspase substrates, the susceptibility of the newly exposed N-terminus of known caspase substrates to protein N-myristoylation was evaluated by in vivo metabolic labeling with [(3)H]myristic acid in transfected cells using a fusion protein in which the query sequence was fused to a model protein. As a result, it was found that the N-terminal nine residues of the newly exposed N-terminus of the caspase-cleavage product of cytoskeletal actin efficiently direct the protein N-myristoylation. Metabolic labeling of COS-1 cells transiently transfected with cDNA coding for full-length truncated actin (tActin) revealed the efficient incorporation of [(3)H]myristic acid into this molecule. When COS-1 cells transiently transfected with cDNA coding for full-length actin were treated with staurosporine, an apoptosis-inducing agent, an N-myristoylated tActin was generated. Immunofluorescence staining coupled with MitoTracker or fluorescence tagged-phalloidin staining revealed that exogenously expressed tActin colocalized with mitochondria without affecting cellular and actin morphology. Taken together, these results demonstrate that the C-terminal 15 kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage during apoptosis and targeted to mitochondria.