Biology subjects

Rowland, A.

Publications and source records attributed to Rowland, A..

3 recordsLinked to original sources

bioRxiv2026

Plastidial Phosphorylase (Pho1a) is the dominant glucosyltransferase regulating starch granule initiation in potato tubers

Starch granule initiation involves the extension of maltooligosaccharide primers by glucosyltransferases. STARCH SYNTHASE 4 (SS4) plays a central role in almost all examined plant species, while the plastidial PHOSPHORYLASE 1 (Pho1) also plays an important role in some species, including rice and wheat. In Arabidopsis, an additional enzymatically inactive homolog of SS4, STARCH SYNTHASE 5 (SS5) contributes to starch granule initiation. To elucidate the mechanism of starch granule initiation in potato tubers, we used CRISPR/Cas9 to generate ss4, ss5, and pho1a knockout mutants in the commercial tetraploid 'Clearwater Russet', to systematically investigate their contribution to granule initiation. In ss4 and ss5 tubers, starch granule size and morphology were unaltered relative to the wild type, suggesting that SS4 and SS5 are dispensable for normal granule initiation in potato tubers. In contrast, pho1a tubers had compound starch granules that arose from multiple initiations, greatly reduced granule size, and highly variable granule morphologies. Affinity pull-down to find Pho1a interaction partners identified LIKE EARLY STARVATION (LESV), although yeast 2-hybrid assays did not show direct protein-protein binding. When expressed alone in Nicotiana benthamiana leaves, Pho1a located to the chloroplast stroma, but when expressed alongside LESV, both proteins co-located on starch granules. This co-localisation, alongside the similar accumulation of small starch granules when LESV is knocked out in tubers, suggest a possible functional interaction in planta. These findings position Pho1a as the central glucosyltransferase in starch granule initiation in Clearwater Russet tubers, where it acts together with LESV.

plant biology↗

bioRxiv2024

CryoEM Structure of the human THIK-1 K2P K+ Channel Reveals a Lower 'Y-gate' Regulated by Lipids and Anaesthetics

THIK-1 (KCNK13) is a halothane-inhibited and anionic lipid-activated Two-Pore Domain (K2P) K+ channel implicated in microglial activation and neuroinflammation, and a current target for the treatment of neurodegenerative disorders such as Alzheimers and Amyothropic Lateral Sclerosis (ALS). However, compared to other K2P channels, little is known about the structural and functional properties of THIK-1. Here we present a 3.16 [A] resolution cryoEM structure of human THIK-1 that reveals several unique features, in particular, a tyrosine in M4 (Y273) which contributes to a lower Y-gate that opens upon activation by physiologically-relevant signalling pathways. We further demonstrate that binding of linoleic acid within a modulatory pocket adjacent to the filter also activates THIK-1, and that halothane inhibition involves a binding site within the inner cavity resulting in changes to the Y-gate. Finally, the extracellular cap domain contains positively-charged residues that line the ion exit pathway and which contribute to the unique biophysical properties of this channel. Overall, our results provide important insights into the structural basis of THIK1 function and identify distinct regulatory sites that expand its potential as a drug target for the modulation of microglial function.

biophysics↗

bioRxiv2022

A metabolite sensor subunit of the Atg1/ULK complex regulates selective autophagy

Cells convert complex metabolic information into stress-adapted autophagy responses. Canonically, multilayered protein kinase networks converge on the conserved Atg1/ULK kinase complex (AKC) to induce non-selective and selective forms of autophagy in response to metabolic changes. Here, we show that, upon phosphate starvation, the metabolite sensor Pho81 interacts with the adaptor subunit Atg11 at the AKC via an Atg11/FIP200 interaction motif to modulate pexophagy by virtue of its conserved phospho-metabolite sensing SPX domain. Notably, we find core AKC components Atg13 and Atg17 are dispensable for phosphate starvation-induced autophagy revealing significant compositional and functional plasticity of the AKC. Our data indicate that, instead of functioning as a selective autophagy receptor, Pho81 compensates for partially inactive Atg13 during pexophagy when TORC1 remains active under phosphate starvation. Our work shows Atg11/FIP200 adaptor subunits not only bind selective autophagy receptors but also modulator subunits that convey metabolic information directly to the AKC for autophagy regulation.

cell biology↗