Role of the Mosaic Cisternal Maturation Machinery in Glycan Synthesis and Oncogenesis

Sahu, Pranoy; Balakrishnan, Aswath; Martino, Rosaria Di; Luini, Alberto; Russo, Domenico

doi:10.3389/fcell.2022.842448

Cited by 2 publications

(2 citation statements)

References 137 publications

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“…Our previous work [23] demonstrated that two of these proteins, B4GALT1 and TGN46 are severely depleted in GARP-KO cells. While most GARP-sensitive proteins localize in trans -Golgi compartments, some of them, like GLG1/MG-160, GALNT1, MAN1A2 [35, 36], are reported to localize in cis/medial Golgi cisternae, indicating that GARP dysfunction is affecting the entire Golgi complex.…”

Section: Resultsmentioning

confidence: 99%

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

Khakurel

Kudlyk

Pokrovskaya

et al. 2022

Preprint

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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 GARP's 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.

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Section: Resultsmentioning

confidence: 99%

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

Khakurel

Kudlyk

Pokrovskaya

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Our previous work (Khakurel et al, 2021) demonstrated that two of these proteins, B4GALT1 and TGN46 are severely depleted in GARP-KO cells. While most GARP-dependent proteins localize in trans-Golgi compartments, some of them, like GLG1/MG-160, GALNT1, MAN1A2 (Gonatas et al, 1989;Sahu et al, 2022), are reported to localize in cis/medial Golgi cisternae, indicating that GARP dysfunction is affecting the entire Golgi complex.…”

Section: Proteomic Analysis Revealed Depletion Of a Subset Of Golgi P...mentioning

confidence: 99%

GARP dysfunction results in COPI displacement, depletion of Golgi v-SNAREs and calcium homeostasis proteins

Khakurel

Kudlyk

Pokrovskaya

et al. 2022

Front. Cell Dev. Biol.

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Golgi-associated retrograde protein (GARP) is an evolutionary conserved heterotetrameric protein complex that tethers endosome-derived vesicles and is vital for Golgi glycosylation. Microscopy and proteomic approaches were employed to investigate defects in Golgi physiology in RPE1 cells depleted for the GARP complex. Both cis and trans-Golgi compartments were significantly enlarged in GARP-knock-out (KO) cells. Proteomic analysis of Golgi-enriched membranes revealed significant depletion of a subset of Golgi residents, including Ca2+ binding proteins, enzymes, and SNAREs. Validation of proteomics studies revealed that SDF4 and ATP2C1, related to Golgi calcium homeostasis, as well as intra-Golgi v-SNAREs GOSR1 and BET1L, were significantly depleted in GARP-KO cells. Finding that GARP-KO is more deleterious to Golgi physiology than deletion of GARP-sensitive v-SNAREs, prompted a detailed investigation of COPI trafficking machinery. We discovered that in GARP-KO cells COPI is significantly displaced from the Golgi and partially relocalized to the ER-Golgi intermediate compartment (ERGIC). Moreover, COPI accessory proteins GOLPH3, ARFGAP1, GBF1, and BIG1 are also relocated to off-Golgi compartments. We propose that the dysregulation of COPI machinery, along with the depletion of Golgi v-SNAREs and alteration of Golgi Ca2+ homeostasis, are the major driving factors for the depletion of Golgi resident proteins, structural alterations, and glycosylation defects in GARP deficient cells.

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Role of the Mosaic Cisternal Maturation Machinery in Glycan Synthesis and Oncogenesis

Cited by 2 publications

References 137 publications

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

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

GARP dysfunction results in COPI displacement, depletion of Golgi v-SNAREs and calcium homeostasis proteins

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