COG5, a subunit of the conserved oligomeric Golgi (COG) complex, plays a critical role in retrograde trafficking within the Golgi apparatus. Dysfunction of COG5 is associated with various human disorders, yet the underlying pathogenic mechanisms remain poorly understood. To investigate the mechanisms, we conducted proteomic analyses using COG5-deficient and rescue cell models, which revealed a potential link between COG5 dysfunction and mitochondrial oxidative phosphorylation (OXPHOS) deficiency. Using COG5-deficient cell models and patient-derived cells harboring COG5 variants, we biochemically validated the involvement of COG5 in mitochondrial OXPHOS, particularly in the regulation of complex I content. These models also exhibited elevated cellular copper levels. Notably, the significant reduction in OXPHOS complexes could be rescued by either restoring COG5 expression or administering a copper chelator. We further demonstrated that excessive cellular copper disrupts the function of mitochondrial iron-sulfur clusters, potentially leading to complex I assembly defects. Additionally, we identified a patient with biallelic COG5 variants presenting with a distinct subtype of mitochondrial disease (Leigh syndrome), a phenotype not previously associated with COG5-related disorders. These findings provide novel mechanistic insights into the role of COG5, extending beyond its established function in Golgi-mediated glycosylation modifications. Our results underscore the importance of COG5 in mitochondrial function through a copper-dependent pathway, offering new perspectives on its contribution to cellular homeostasis and disease pathogenesis.

Salt stress severely restricts plant growth and agricultural productivity. The Conserved Oligomeric Golgi complex (COG) plays a key role in vesicular trafficking, but its function in plant stress responses remains poorly understood. Here, we demonstrated that the expression of the subunit COG3 of the COG complex in Arabidopsis thaliana was significantly induced under salt stress. The COG3 knockdown mutant (cog3) exhibits severe growth and development defects, as well as high sensitivity to salt stress. Furthermore, genetic complementation with COG3-GFP partially rescued the salt-sensitive phenotype of the cog3 mutant, while overexpression of COG3 enhanced plant salt tolerance. Mechanistically, COG3 physically interacts with the Sorting Nexin 1 (SNX1) on the endosome, and genetic evidence indicates that COG3 acts upstream of SNX1. We propose that under salt stress, COG3 maintains Golgi structural integrity and stabilizes SNX1 protein, thereby facilitating SNX1-mediated recycling of cargo proteins from endosomes to the plasma membrane to maintain cellular ion homeostasis. Loss of COG3 function disrupts this recycling pathway, leading to salt hypersensitivity. Our study reveals a novel COG3-SNX1 regulatory module that mediates protein recycling to enhance salt tolerance, providing new insights into the molecular mechanisms underlying plant adaptation to abiotic stress.

The endoplasmic reticulum-Golgi intermediate compartment (ERGIC) plays a crucial role in the secretory pathway; however, its existence and function in lower eukaryotes remain largely unexamined. In this study, we identified Emp43 (SPBC4F6.05c) of Schizosaccharomyces pombe, an orthologue of human (Homo sapiens) ERGIC-53, and demonstrated its localization to an ERGIC-like compartment. The localization of Emp43 depended on its C-terminal KYL motif and oligomerization through the CC1 domain. Deletion of S. pombe emp43+ resulted in significant sensitivity to MgCl2 and FK506, along with defects in septum integrity, indicating a role in cell wall maintenance. Further analysis identified Ssp120 of S. pombe, an orthologue of human MCFD2, as a functional partner of Emp43. Yeast two-hybrid assays confirmed a strong interaction between Emp43 and Ssp120, and both proteins co-localized within an ERGIC-like compartment. Additionally, we identified Meu17 of S. pombe, a glucan-α-1,4-glucosidase homolog, as a potential ligand for Emp43. Overexpression of Meu17 rescued MgCl2 sensitivity in both emp43Δ and ssp120Δ strains, while mutations in its N-linked glycosylation sites (N383, N409) or its predicted active site (D203) disrupted its septum localization and functional rescue capability. Our findings indicate that Emp43 forms a complex with Ssp120 to facilitate the transport of glycosylated proteins, such as Meu17, within an ERGIC-like compartment in fission yeast S. pombe. This study provides the first evidence of an ERGIC-like structure in S. pombe and highlights the conserved nature of ERGIC-associated mechanisms across eukaryotes.

AB toxins are a diverse family of protein toxins that enter host cells via endocytosis and induce cell death. In yeast, the AB toxin K28 is internalised to endosomes of susceptible yeast, before following the retrograde trafficking pathway and ultimately triggering cell cycle arrest. The endolysosomal defence factor Ktd1 protects against K28, but its regulation remains unclear. We show all lobe B subunits of the conserved oligomeric Golgi (COG) tethering complex are required for K28 resistance. Our experiments suggest the hypersensitivity of cog mutants is primarily explained by defects in Ktd1 trafficking. Ktd1 mis-localisation in cog mutants is reminiscent of disruptions in Snc1, a surface cargo that recycles multiple times via the Golgi. This work suggests not only that the COG complex is responsible for the precise trafficking of Ktd1 required to mediate toxin defence, but that Ktd1 might survey endolysosomal compartments for toxin. This work underpins the importance of Ktd1 in defence against the AB toxin K28, and implies how various membrane trafficking regulators could influence toxin effects in other eukaryotic systems.

Intracellular trafficking relies on small membrane intermediates that transport cargo between different compartments. However, the precise role of vesicles in preserving Golgi function remains uncertain. To clarify this, we induced acute inactivation of the Conserved Oligomeric Golgi (COG) complex and analyzed vesicles from the different Golgi compartments. Proteomic analysis of the resulting vesicles revealed distinct molecular profiles, indicating a robust recycling system for Golgi proteins. All glycosylation enzymes and sugar transporters were detected in immunoisolated vesicles. The abundance of glycosylation machinery in intra-Golgi vesicles significantly increased following acute COG malfunction. Vesicles isolated from wild-type cells retained various vesicular coats, which were detaching from COG complex-dependent (CCD) vesicles stalled in the untethered state. Additionally, COG depletion led to increased molecular overlap among different populations of vesicles, suggesting that defects in vesicle tethering disrupt intra-Golgi sorting. Notably, CCD vesicles were functional and could be specifically rerouted to mitochondria that ectopically express Golgi tethers. Our findings demonstrate that the entire Golgi glycosylation machinery recycles within vesicles in a COG-dependent manner, whereas secretory and ER-Golgi trafficking proteins were not enriched. These results support a model in which the COG complex orchestrates the multistep recycling of glycosylation machinery, coordinated by specific coats, tethers, and SNAREs.