The Conserved N-Terminal Extension of AtKEA1 Is Largely Dispensable for Plastid Function but Contributes to Potassium Homeostasis

Functional and transcriptomic analyses in Neurospora crassa reveal a crucial role for the N-glycoprotein deglycosylation process in fungal homeostasis.

N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, efficient endoplasmic reticulum (ER) quality control, such as that provided by the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-β-n-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the cytosolic ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-β-n-acetylhexosaminidase under ER stress. These findings identify the cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.

OEF18 is a membrane-anchored organellar Ca²⁺ sensor linking calcium signaling to jasmonate-mediated defense and stress acclimation

The Rab7 C-terminal intrinsically disordered region is a key determinant of development and pathogenicity in Aspergillus flavus.

Rab7 GTPases coordinate late endosome maturation, vesicle trafficking, and vacuole biogenesis across eukaryotes, yet the functional contribution of the Rab7 C-terminal tail to organismal physiology remains incompletely defined. Here we present the crystal structure of Aspergillus flavus Rab7 (AflRab7), which reveals a well-ordered GTPase core, whereas its C-terminal tail is conserved across Aspergillus species yet lacks interpretable electron density, consistent with an intrinsically disordered region (IDR). Biochemical assays show that deletion of this IDR does not alter basal GTP hydrolysis, whereas genetic and cell-biological studies demonstrate that deletion of residues 191-205 causes severe growth retardation, defective conidiation and sclerotium formation, markedly reduces aflatoxin production, and fragmented vacuolar compartments. The IDR-deficient mutants further exhibit pronounced hypersensitivity to cell-wall stress and altered tolerance to membrane, osmotic, and redox challenges. Transcriptome profiling, supported by qPCR validation, reveals extensive transcriptional reprogramming upon loss of the C-terminal region, including dysregulation of pathways linked to development and secondary metabolism. Overall, our findings extend the mechanistic understanding of Rab7 beyond its catalytic pocket and establish the AflRab7 C-terminal IDR as a critical regulatory element that couples vacuolar dynamics to A. flavus development and aflatoxin biosynthesis, highlighting a tractable target for mitigating fungal toxigenic potential.

The PHO1 protein family in phosphate export and beyond

Abstract The Arabidopsis thaliana PHOSPHATE 1 (PHO1) was first identified as an inorganic phosphate (Pi) exporter mediating Pi loading into the root xylem. PHO1 is the prototypical member of a family of eukaryotic proteins possessing both an SPX and EXS domain. Recent cryo-EM structures of both the Arabidopsis PHO1 homolog PHO1;H1 and its mammalian ortholog Xenotropic and Polytropic Retrovirus Receptor 1 (XPR1), revealed that these proteins work as Pi channels that use a double-gated mechanism controlled by inositol polyphosphate. Key amino acids involved in binding Pi and gating the channel are highly conserved in PHO1 homolog throughout eukaryote superphyla, including in unicellular organisms, indicating a possible conservation of function during evolution. Beyond their structural homology, both plant and animal PHO1 ortholog are primarily localized to intracellular vesicles, raising interesting questions about the cell biology of Pi export. Arabidopsis PHO1 also contributes to the response of guard cells and seeds to abscisic acid (ABA) through yet unknown mechanisms. In this review, we will highlight how the recently gained knowledge about PHO1 and XPR1 structure and its cell biology is shaping our views about how these proteins contribute to Pi homeostasis at the cellular and organismal levels.

Discovery of a regulatory node that coordinates cell envelope assembly in mycobacteria

Mycobacteria possess a complex double-membrane cell envelope critical for survival and pathogenesis. Proper assembly of this architecture requires the biosynthesis and transport of major components, including arabinogalactan (AG) polysaccharides and mycolic acids (MAs), but how these processes are effectively coordinated is unknown. Here, we discover an essential membrane complex that serves as a regulatory node in mycobacterial envelope biogenesis. The acyltransferase TmaT and the arabinofuranosyltransferase AftD physically interact; cryo-EM structures reveal a 1:1 stoichiometry, and present a novel fold for TmaT, featuring a central channel that binds co-factor for acetylation in the periplasm. We establish that the TmaT-AftD interaction, and the catalytic activities of both enzymes, are required for MA transport across the cell envelope, as well as AG ligation to the cell wall, the final stage of AG biosynthesis. The TmaT-AftD complex coordinates the two major envelope assembly pathways, presenting a structural vulnerability for future anti-mycobacterial drug development.

A FtsH-Like Zinc Metalloprotease, OsFtsH1, Coordinates Chloroplast Development and Photosystem II Homeostasis in Rice.

Chloroplast development is a fundamental process underlying photosynthesis and plant growth, yet its molecular regulatory mechanisms remain to be fully elucidated. In this study, we identified an albino rice mutant, which exhibits drastically reduced chlorophyll content and defective chloroplast ultrastructure. Through map-based cloning, coupled with CRISPR/Cas9-mediated gene editing and complementation assays, we verified that the target gene encodes a zinc metalloprotease of the FtsH protein family, OsFtsH1. The expression of OsFtsH1 is light-inducible, and its encoded protein localizes specifically to chloroplasts. RNA-sequencing (RNA-seq) analysis revealed broad differential expression of photosynthesis-related genes in osftsh1 mutants. qRT-PCR further demonstrated that plastid-encoded genes involved in chloroplast biogenesis are markedly downregulated at the transcriptional level. Functional assays indicated that OsFtsH1 sustains the homeostasis of D1, D2 and CP43, core proteins of the photosystem II (PSII), and collaboratively regulates chloroplast development through interactions with three key proteins: the chloroplast signaling protein OsCPL1, the PSII oxygen-evolving complex component OsPsbO, and the photosynthetic electron transport protein OsFd1. Additionally, the expression of OsFtsH1 is induced by indole-3-acetic acid (IAA) and abscisic acid (ABA), and its overexpression markedly enhances rice sensitivity to these two phytohormones. Collectively, our findings unravel the multifaceted and crucial functions of OsFtsH1 in orchestrating chloroplast development, photosynthetic machinery homeostasis, and stress response in rice.