Characterization of two distinct binding modes between syntaxin 4 and Munc18c

Interaction of SM (Sec1/Munc18) proteins with their cognate syntaxins represents an important regulatory mechanism of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)-mediated membrane fusion. Understanding the conserved mechanisms by which SM proteins function in this process has proved challenging, largely due to an apparent lack of conservation of binding mechanisms between different SM-syntaxin pairs. In the present study, we have identified a hitherto uncharacterized mode of binding between syntaxin 4 and Munc18c that is independent of the binding mode shown previously to utilize the N-terminal peptide of syntaxin 4. Our data demonstrate that syntaxin 4 and Munc18c interact via two distinct modes of binding, analogous to those employed by syntaxin 1a-Munc18a and syntaxin 16-Vps45p (vacuolar protein sorting 45). These data support the notion that all syntaxin/SM proteins bind using conserved mechanisms, and pave the way for the formulation of unifying hypotheses of SM protein functio

Regulations of RNA Polymerase III transcription by Maf1 in mammalian cells

RNA polymerase (pol) III produces essential components of the biosynthetic machinery; therefore, its output is tightly coupled with the rate of cell growth and proliferation. In <i>Saccharomyces cerevisiae</i>, Maf1 is an essential mediator of pol III repression in response to starvation. We demonstrate that a Maf1 ortholog is also used to restrain pol III activity in mouse and human cells. Mammalian Maf1 represses pol III transcription <i>in vitro</i> and in transfected fibroblasts. Furthermore, genetic deletion of Maf1 elevates pol III transcript expression, thus confirming the role of endogenous Maf1 as an inhibitor of mammalian pol III output. Maf1 is detected at chromosomal pol III templates in rodent and human cells. It interacts with pol III as well as its associated initiation factor TFIIIB and is phosphorylated in a serum-sensitive manner <i>in vivo</i>. These aspects of Maf1 function have been conserved between yeast and mammals and are therefore likely to be of fundamental importance in controlling pol III transcriptional activit

Autoimmune disease-associated histamine receptor H1 alleles exhibit differential protein trafficking and cell surface expression

Structural polymorphisms (L263P, M313V, and S331P) in the third intracellular loop of the murine histamine receptor H1 (H1R) are candidates for Bphs, a shared autoimmune disease locus in experimental allergic encephalomyelitis and experimental allergic orchitis. The P-V-P haplotype is associated with increased disease susceptibility (H1RS) whereas the L-M-S haplotype is associated with less severe disease (H1RR). In this study, we show that selective re-expression of the H1RS allele in T cells fully complements experimental allergic encephalomyelitis susceptibility and the production of disease-associated cytokines while selective re-expression of the H1RR allele does not. Mechanistically, we show that the two H1R alleles exhibit differential cell surface expression and altered intracellular trafficking, with the H1RR allele being retained within the endoplasmic reticulum. Moreover, we show that all three residues (L-M-S) comprising the H1RR haplotype are required for altered expression. These data are the first to demonstrate that structural polymorphisms influencing cell surface expression of a G protein-coupled receptor in T cells regulates immune functions and autoimmune disease susceptibility

mTOR associates with TFIIIC, is found at tRNA and 5S rRNA genes, and targets their repressor Maf1

Synthesis of tRNA and 5S rRNA by RNA polymerase (pol) III is regulated by the mTOR pathway in mammalian cells. The mTOR kinase localizes to tRNA and 5S rRNA genes, providing an opportunity for direct control. Its presence at these sites can be explained by interaction with TFIIIC, a DNA-binding factor that recognizes the promoters of these genes. TFIIIC contains a TOR signaling motif that facilitates its association with mTOR. Maf1, a repressor that binds and inhibits pol III, is phosphorylated in a mTOR-dependent manner both in vitro and in vivo at serine 75, a site that contributes to its function as a transcriptional inhibitor. Proximity ligation assays confirm the interaction of mTOR with Maf1 and TFIIIC in nuclei. In contrast to Maf1 regulation in yeast, no evidence is found for nuclear export of Maf1 in response to mTOR signaling in HeLa cells. We conclude that mTOR associates with TFIIIC, is recruited to pol III-transcribed genes, and relieves their repression by Maf

Human La is found at RNA polymerase III-transcribed genes in vivo

The human La autoantigen can bind to nascent RNA transcripts and has also been postulated to act as an RNA polymerase III (pol III) transcription initiation and termination factor. Here, we show by chromatin immunoprecipitation (ChIP) that La is associated with pol III-transcribed genes in vivo. In contrast, the Ro autoantigen, which can also bind pol III transcripts, is not found at these genes. The putative pol III transcription factors NF1 and TFIIA are also not detected at class III genes. Binding of La remains when transcription is repressed at mitosis and does not correlate with the presence of polymerase at the gene. However, gene occupancy depends on the phosphorylation status of La, with the less prevalent, unphosphorylated form being found selectively on pol III templates