Biology subjects

Pokrovskaya, I.

Publications and source records attributed to Pokrovskaya, I..

3 recordsLinked to original sources

bioRxiv2026

Defining the landscape of the Golgi CATCHRs

Approximately one-third of all human proteins transit through the secretory pathway, where the Golgi apparatus orchestrates protein modification, sorting, and distribution through highly selective vesicle budding and fusion events. Central to these processes are the Complexes Associated with Tethering Containing Helical Rods (CATCHR), multisubunit tethering complexes that coordinate vesicle docking and fusion through interactions with coiled-coil tethers (CCTs), Rab GTPases, SNAREs, and Sec1/Munc18 (SM) proteins and other trafficking factors. To define the molecular organization of Golgi CATCHR complexes, we generated the first comprehensive proximity-interaction map of the COG, GARP, and EARP tethering complexes using functional, near-endogenously expressed TurboID-tagged subunits. Comparative proximity proteomics revealed that each CATCHR complex assembles a distinct trafficking module composed of characteristic CCTs, Rab-associated proteins, SNAREs, and SM proteins, establishing a system-level framework for the spatial organization of Golgi and endosomal membrane trafficking. The COG complex preferentially associated with Golgi CCTs and the STX5-SCFD1 fusion machinery, GARP with CCDC186, and STX16-VPS45 pathway, and EARP with GRIPAP1, the VPS33B-VIPAS39 (CHEVI) complex, and RAB11-dependent recycling machinery. Beyond validating known interactions, our study identifies CCDC186 as a vesicle tether, establishes WWOX as a previously unrecognized regulator of Golgi homeostasis and glycosylation, and provides evidence that Golgi CATCHR complexes function as central organizing hubs that assemble specialized trafficking modules to coordinate vesicle tethering and membrane fusion.

Cell Biology↗

bioRxiv2024

Acute GARP depletion disrupts vesicle transport leading to severe defects in sorting, secretion, and O-glycosylation

The GARP complex is an evolutionarily conserved protein complex proposed to tether endosome-derived vesicles at the trans-Golgi network. While prolonged depletion of GARP leads to severe trafficking and glycosylation defects, the primary defects linked to GARP dysfunction remain unclear. In this study, we utilized the mAID degron strategy to achieve rapid degradation of VPS54 in human cells, acutely disrupting GARP function. This resulted in the partial mislocalization and degradation of a subset of Golgi-resident proteins, including TGN46, ATP7A, TMEM87A, CPD, C1GALT1, and GS15. Enzyme recycling defects led to the early onset of O-glycosylation abnormalities. Additionally, while the secretion of fibronectin and cathepsin D was altered, mannose-6-phosphate receptors were largely unaffected. Partial displacement of COPI, AP1, and GGA coats caused a significant accumulation of vesicle-like structures and large vacuoles. Electron microscopy detection of GARP-dependent vesicles, along with the identification of specific cargo proteins, provides direct experimental evidence of GARPs role as a vesicular tether. We conclude that the primary defects of GARP dysfunction involve vesicular coat mislocalization, accumulation of GARP-dependent vesicles, degradation and mislocalization of specific Golgi proteins, and O-glycosylation defects.

cell biology↗

bioRxiv2022

GARP complex controls Golgi physiology by stabilizing COPI machinery and Golgi v-SNAREs

GARP is an evolutionary conserved heterotetrameric protein complex that is thought to tether endosome-derived vesicles and promotes their fusion in the trans-Golgi network. We have previously discovered the GARPs role in maintaining Golgi glycosylation machinery. To further investigate the importance of the GARP complex for Golgi physiology, we employed Airyscan superresolution and electron microscopy, as well as the unbiased quantitative proteomic analysis of Golgi in RPE1 cells. Both cis and trans-Golgi compartments were significantly enlarged in GARP deficient cells with pronounced alterations of TGN morphology. In GARP-KO cells, proteomic analysis revealed a depletion of a subset of Golgi resident proteins, including Ca2+ binding proteins, glycosylation enzymes, and v-SNAREs. We validated proteomics studies and discovered that two Golgi-resident proteins SDF4 and ATP2C1, related to Golgi calcium homeostasis, as well as intra-Golgi v-SNAREs GOSR1 and BET1L, are significantly depleted in GARP-KO cells. To test if SNARE depletion is responsible for the Golgi defects in GARP deficient cells, we created and analyzed GOSR1 and BET1L KO cell lines. Since GARP-KO was more deleterious to the Golgi physiology than SNARE-KOs, we have investigated other components of intra-Golgi vesicular trafficking, particularly COPI vesicular coat and its accessory proteins. We found that COPI is partially relocalized to the ERGIC compartment in GARP-KO cells. Moreover, COPI accessory proteins GOLPH3, ARFGAP1, GBF1 were displaced from the membrane and BIG1 was relocated to endolysosomal compartment in GARP-KO cells. We propose that the dysregulation of COPI machinery along with degradation of intra-Golgi v-SNAREs and alteration of Golgi Ca2+ homeostasis are the major driving factors for the instability of Golgi resident proteins and glycosylation defects in GARP deficient cells.

cell biology↗