Biology subjects

Sekiguchi, Y.

Publications and source records attributed to Sekiguchi, Y..

3 recordsLinked to original sources

bioRxiv2026

Improved adenine-HPLC method for quantifying yeast based on cellular DNA content

Accurate quantification of fungi is important for a myriad of applications but remains challenging. Previously, we demonstrated that an approach called the adenine-HPLC method can quantify bacteria, including those with aggregating properties that are difficult to quantify using conventional methods, by measuring cellular adenine derived from DNA and converting the adenine amount to genome copy number, without being influenced by cell morphology. However, in this study, when this adenine-HPLC method was applied to the quantification of budding yeast as a model fungus, accurate measurement proved impossible. This limitation was attributed to adenine release from other adenine-containing biomolecules, such as RNA and ATP, and we therefore developed a method that suppresses adenine release from these molecules. This method involves reducing the temperature of the acid treatment and prewashing the cells before acid treatment. In addition, we incorporated a process that corrects for the naturally occurring free adenine level as background during total adenine measurement. The improved adenine-HPLC method based on these modifications enables accurate quantification of budding yeast using genomic DNA content in whole cells as the quantification unit.

microbiology↗

bioRxiv2026

Cryo-EM structures of SAMD9L reveal the arrangement and coordination of multi-domains

Human sterile alpha motif domain-containing 9 (hSAMD9L) is a large ([~]185 kDa) multi-domain interferon-stimulated antiviral effector with strong translation-inhibitory activity. Inherited heterozygous gain-of-function (GoF) mutations in SAMD9L directly associated with severe bone marrow failure syndromes. Using single-particle cryo-electron microscopy (cryo-EM), we determined the first structures of both full-length wild-type hSAMD9L and an N-terminal-truncated mutant at resolutions ranging from 2.8 to 3.7 [A]. Both proteins exist in monomeric and dimeric states, providing clear evidence that the sterile alpha motif (SAM) and AlbA domains are not essential for dimerization. Our cryo-EM analysis reveals a tightly packed, closed architecture defined by interlocking multi-domains. We precisely mapped the extensive dimer interface mediated by Sir2-like and an oligonucleotide/oligosaccharide-binding (OB) domains. Biochemical analyses show that hSAMD9L binds double-stranded DNA in vitro but has no detectable NTP hydrolysis activity under our assay conditions. Accordingly, in our cryo-EM map we observed a clear density for a non-hydrolyzed NTP in the pocket, suggesting nucleotide binding without turnover like in STAND (Signal Transduction ATPases with Numerous Domains) proteins. Together, these results provide a structural framework for hSAMD9L and also providing key insights into its organization, domain packing and dimerization and offer a basis for understanding how GoF variants may alter hSAMD9L regulation thus impacting cell proliferation.

biochemistry↗

bioRxiv2024

SLC35G1: A highly chloride-sensitive transporter responsible for the basolateral membrane transport in intestinal citrate absorption

The intestinal absorption of essential nutrients, especially those not readily biosynthesized, is a critical physiological process for maintaining homeostasis. Numerous studies have indicated that intestinal absorption is mediated by various membrane transporters. Citrate, a crucial bioactive compound produced as an intermediate in the Krebs cycle, is absorbed in the small intestine through carrier-mediated systems because of its high hydrophilicity. While the luminal absorption of citrate is mediated by Na+-dicarboxylate cotransporter 1 (NaDC1/SLC13A2), the mechanism governing the release of the transported citrate into the bloodstream remains unknown. Here, we explored the transporters responsible for intestinal citrate absorption at the basolateral membrane, focusing on highly expressed orphan transporters in the small intestine as candidates. Consequently, SLC35G1, originally identified as a partner of stromal interaction molecule 1, a cell surface transmembrane glycoprotein, was found to play a role in the intestinal absorption of citrate at the basolateral membrane. Furthermore, our results revealed that SLC35G1-mediated citrate transport was diminished by chloride ions at physiologically relevant extracellular concentrations. This suggests that SLC35G1, to our best knowledge, is the first transporter identified to be extremely sensitive to chloride ions among those functioning on the basolateral membrane of intestinal epithelial cells. This study provides valuable insights into the intestinal absorption of citrate and significantly contributes to elucidating the poorly understood molecular basis of the intestinal absorption system.

physiology↗