Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine-N2-) Methyltransferase (Trm11-Trm112 Complex)

The Saccharomyces cerevisiae Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms N2-methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112. Furthermore, the CCA terminus is essential for methylation by Trm11-Trm112, and Trm11-Trm112 also only methylates tRNAs with a regular-size variable region. In addition, the G10-C25 base pair is required for methylation by Trm11-Trm112. The data also demonstrated that Trm11-Trm112 recognizes the anticodon-loop and that U38 in tRNAAla acts negatively in terms of methylation. Likewise, the U32-A38 base pair in tRNACys negatively affects methylation. The only exception in our in vitro study was tRNAValAAC1. Our experiments showed that the tRNAValAAC1 transcript was slowly methylated by Trm11-Trm112. However, position 10 in this tRNA was reported to be unmodified G. We purified tRNAValAAC1 from wild-type and trm11 gene deletion strains and confirmed that a portion of tRNAValAAC1 is methylated by Trm11-Trm112 in S. cerevisiae. Thus, our study explains the m2G10 modification pattern of all S. cerevisiae class I tRNAs and elucidates the Trm11-Trm112 binding sites

Spatiotemporal Analysis of the Molecular Interaction between PICK1 and PKC

PICK1 is a protein which was initially identified as a protein kinase Cα (αPKC) binding protein using the yeast two-hybrid system. In addition to αPKC, the PICK1 complex binds to and regulates various transmembrane proteins including receptors and transporters. However, it has not been clarified when and where PICK1 binds to αPKC. We examined the spatio­temporal interaction of PICK1 and PKC using live imaging techniques and showed that the activated αPKC binds to PICK1 and transports it to the plasma membrane. Although the membrane translocation of PICK1 requires the activation of αPKC, PICK1 is retained on the membrane even after PKC moves back to the cytosol. These results suggest that the interaction between αPKC and PICK1 is transient and may not be necessary for the regulation of receptors/transporters by PICK1 or by αPKC on the membrane

Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine-<i>N</i>²-) Methyltransferase (Trm11-Trm112 Complex)

The Saccharomyces cerevisiae Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms N2-methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112. Furthermore, the CCA terminus is essential for methylation by Trm11-Trm112, and Trm11-Trm112 also only methylates tRNAs with a regular-size variable region. In addition, the G10-C25 base pair is required for methylation by Trm11-Trm112. The data also demonstrated that Trm11-Trm112 recognizes the anticodon-loop and that U38 in tRNAAla acts negatively in terms of methylation. Likewise, the U32-A38 base pair in tRNACys negatively affects methylation. The only exception in our in vitro study was tRNAValAAC1. Our experiments showed that the tRNAValAAC1 transcript was slowly methylated by Trm11-Trm112. However, position 10 in this tRNA was reported to be unmodified G. We purified tRNAValAAC1 from wild-type and trm11 gene deletion strains and confirmed that a portion of tRNAValAAC1 is methylated by Trm11-Trm112 in S. cerevisiae. Thus, our study explains the m2G10 modification pattern of all S. cerevisiae class I tRNAs and elucidates the Trm11-Trm112 binding sites

Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine-N2-) Methyltransferase (Trm11-Trm112 Complex)

The Saccharomyces cerevisiae Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms N2-methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112. Furthermore, the CCA terminus is essential for methylation by Trm11-Trm112, and Trm11-Trm112 also only methylates tRNAs with a regular-size variable region. In addition, the G10-C25 base pair is required for methylation by Trm11-Trm112. The data also demonstrated that Trm11-Trm112 recognizes the anticodon-loop and that U38 in tRNAAla acts negatively in terms of methylation. Likewise, the U32-A38 base pair in tRNACys negatively affects methylation. The only exception in our in vitro study was tRNAValAAC1. Our experiments showed that the tRNAValAAC1 transcript was slowly methylated by Trm11-Trm112. However, position 10 in this tRNA was reported to be unmodified G. We purified tRNAValAAC1 from wild-type and trm11 gene deletion strains and confirmed that a portion of tRNAValAAC1 is methylated by Trm11-Trm112 in S. cerevisiae. Thus, our study explains the m2G10 modification pattern of all S. cerevisiae class I tRNAs and elucidates the Trm11-Trm112 binding sites

Immunogold Electron Microscopic Demonstration of Distinct Submembranous Localization of the Activated γPKC Depending on the Stimulation

We examined the precise intracellular translocation of γ subtype of protein kinase C (γPKC) after various extracellular stimuli using confocal laser-scanning fluorescent microscopy (CLSM) and immunogold electron microscopy. By CLSM, treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a slow and irreversible accumulation of green fluorescent protein (GFP)-tagged γPKC (γPKC–GFP) on the plasma membrane. In contrast, treatment with Ca2+ ionophore and activation of purinergic or NMDA receptors induced a rapid and transient membrane translocation of γPKC–GFP. Although each stimulus resulted in PKC localization at the plasma membrane, electron microscopy revealed that γPKC showed a subtle but significantly different localization depending on stimulation. Whereas TPA and UTP induced a sustained localization of γPKC–GFP on the plasma membrane, Ca2+ ionophore and NMDA rapidly translocated γPKC–GFP to the plasma membrane and then restricted γPKC–GFP in submembranous area (<500 nm from the plasma membrane). These results suggest that Ca2+ influx alone induced the association of γPKC with the plasma membrane for only a moment and then located this enzyme at a proper distance in a touch-and-go manner, whereas diacylglycerol or TPA tightly anchored this enzyme on the plasma membrane. The distinct subcellular targeting of γPKC in response to various stimuli suggests a novel mechanism for PKC activation. (J Histochem Cytochem 56:253–265, 2008