Interacting partners of Golgi‐localized small G protein Arl5b identified by a combination of <i>in vivo</i> proximity labelling and GFP‐Trap pull down

Houghton, Fiona; Makhoul, Christian; Cho, Ellie Hyun-Jung; Williamson, Nicholas A.; Gleeson, Paul A.

doi:10.1002/1873-3468.14443

Cited by 5 publications

(7 citation statements)

References 41 publications

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“…We performed highresolution Airyscan imaging of HeLa cells immunolabeled for ACBD3 and cis and trans-Golgi markers. Despite having no transmembrane domain or other known Golgi interacting domain 32 , ACBD3 showed significant colocalisation with cis and trans-Golgi markers (Figure S1A and B), consistent with previous findings 35,36 . To identify the domain by which ACBD3 is recruited to the Golgi apparatus, we examined other known ACBD3 recruitment factors.…”

Section: Acbd3 Is Recruited To the Golgi Apparatus Via A Proteinprote...supporting

confidence: 91%

“…Our data and others demonstrate that although we can recapitulate an interaction with giantin, it is not solely responsible for the recruitment of ACBD3 to the Golgi apparatus. ACBD3 has also been proposed to be recruited to the Golgi apparatus by ARL5B 35 . Loss of ARL5B caused 50% of ACBD3 Golgi localisation to be lost.…”

Section: Discussionmentioning

confidence: 99%

“…Initially, based on a yeast-2 hybrid interaction, the golgin giantin was proposed to recruit ACBD3 32 ; however, the knock-down of giantin does not result in the loss of ACBD3 recruitment to the Golgi apparatus 29 . Recently, the loss of ARL5B was demonstrated to cause the loss of the Golgi pool of ACBD3 35 ; however, ARL5B is trans-Golgi localised compared to the cis-and trans-Golgi localised ACBD3. Therefore, the mechanism by which ACBD3 is recruited to the Golgi apparatus remains unclear.…”

Section: Introductionmentioning

confidence: 99%

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Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

Stalder

Yakunin

Gershlick

2023

Preprint

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ACBD3 is a protein localised to the Golgi apparatus and recruits other proteins, such as PI4KIIIβ, to the Golgi. However, the mechanism through which ACBD3 itself is recruited to the Golgi is poorly understood. This study demonstrates there are two mechanisms for ACBD3 recruitment to the Golgi. First, we identified that an MWT374-376motif in the unique region upstream of the GOLD domain in ACBD3 is essential for Golgi localisation. Second, we use unbiased proteomics to demonstrate that ACBD3 interacts with SCFD1, a Sec1/Munc-18 (SM) protein, and a SNARE protein, SEC22B. CRISPR-KO of SCFD1 causes ACBD3 to become cytosolic. We also found that ACBD3 is redundantly recruited to the Golgi apparatus by two golgins: golgin-45 and giantin, which bind to ACBD3 through interaction with the MWT374-376motif. Taken together, our results demonstrate that ACBD3 is recruited to the Golgi in a two-step sequential process, with the SCFD1-mediated interaction occurring upstream of the interaction with the golgins.

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Section: Acbd3 Is Recruited To the Golgi Apparatus Via A Proteinprote...supporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

Stalder

Yakunin

Gershlick

2023

Preprint

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“…, 2021 ). However, BioID labeling efficiency will determine which potential partners are identified, and studies from our lab comparing proximity labeling and pull downs of the same bait (Arl5b) showed that the pull downs detected proteins that were not detected in the proximity labeling procedures ( Houghton et al. , 2022 ).…”

Section: Discussionmentioning

confidence: 99%

Arf5-mediated regulation of mTORC1 at the plasma membrane

Makhoul¹,

Houghton²,

Hinde

et al. 2023

MBoC

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The mechanistic target of rapamycin (mTOR) kinase regulates a major signalling pathway in eukaryotic cells. In addition to regulation of mTORC1 at lysosomes, mTORC1 is also localised at other locations. However, little is known about the recruitment and activation of mTORC1 at non-lysosomal sites. To identify regulators of mTORC1 recruitment to non-lysosomal compartments, novel interacting partners with the mTORC1 subunit, Raptor, were identified using immunoprecipitation and mass spectrometry. We show that one of the interacting partners, Arf5, is a novel regulator of mTORC1 signalling at plasma membrane ruffles. Arf5-GFP localizes with endogenous mTOR at PI3,4P2 enriched membrane ruffles together the GTPase required for mTORC1 activation, Rheb. Knockdown of Arf5 reduced the recruitment of mTOR to membrane ruffles. The activation of mTORC1 at membrane ruffles was directly demonstrated using a plasma membrane-targeted mTORC1 biosensor and Arf5 shown to enhance the phosphorylation of the mTORC1 biosensor substrate. In addition, endogenous Arf5 was shown to be required for rapid activation of mTORC1-mediated S6 phosphorylation following nutrient starvation and re-feeding. Our findings reveal a novel Arf5-dependent pathway for recruitment and activation of mTORC1 at plasma membrane ruffles, a process relevant for spatial and temporal regulation of mTORC1 by receptor and nutrient stimuli.

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“…, 2017 ). Recently, the loss of ARL5B was demonstrated to cause the loss of the Golgi pool of ACBD3 ( Houghton et al. , 2022 ); however, ARL5B is trans -Golgi localized compared with the cis - and trans -Golgi localized ACBD3.…”

Section: Introductionmentioning

confidence: 99%

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

Stalder,

Yakunin,

Pereira

et al. 2024

MBoC

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ACBD3 is a protein localised to the Golgi apparatus and recruits other proteins, such as PI4KIIIβ, to the Golgi. However, the mechanism through which ACBD3 itself is recruited to the Golgi is poorly understood. This study demonstrates there are two mechanisms for ACBD3 recruitment to the Golgi. First, we identified that an MWT374-376 motif in the unique region upstream of the GOLD domain in ACBD3 is essential for Golgi localisation. Second, we use unbiased proteomics to demonstrate that ACBD3 interacts with SCFD1, a Sec1/Munc-18 (SM) protein, and a SNARE protein, SEC22B. CRISPR-KO of SCFD1 causes ACBD3 to become cytosolic. We also found that ACBD3 is redundantly recruited to the Golgi apparatus by two golgins: golgin-45 and giantin, which bind to ACBD3 through interaction with the MWT374-376 motif. Taken together, our results suggest that ACBD3 is recruited to the Golgi in a two-step sequential process, with the SCFD1-mediated interaction occurring upstream of the interaction with the golgins.

show abstract

Interacting partners of Golgi‐localized small G protein Arl5b identified by a combination of in vivo proximity labelling and GFP‐Trap pull down

Cited by 5 publications

References 41 publications

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

Arf5-mediated regulation of mTORC1 at the plasma membrane

Recruitment of PI4KIIIβ to the Golgi by ACBD3 is dependent on an upstream pathway of a SNARE complex and golgins

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