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The FAM114A proteins are adaptors for the recycling of Golgi enzymes - Fig1E-F

ABSTRACT: The Golgi apparatus contains many resident enzymes that must remain in place whilst their substrates flow through on their journey from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the rims of the Golgi stack to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and at least one COPI adaptor protein, GOLPH3, has been shown to recruit enzymes into vesicles in a specific part of the stack. We have used proximity biotinylation to identify further components of intra-Golgi transport vesicles and found FAM114A2, an uncharacterised cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1 showed that they bind to numerous Golgi resident proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not result in substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Generation of Drosophila mutants lacking FAM114A revealed defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins are COPI vesicle resident proteins that bind to Golgi enzymes and so are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Cicer Arietinum (chickpea) (garbanzo) Danio Rerio (zebrafish) (brachydanio Rerio) Drosophila Melanogaster (fruit Fly) Dictyostelium Discoideum (slime Mold)

SUBMITTER: Catarina Franco

LAB HEAD: Sean Munro

PROVIDER: PXD054187 | Pride | 2024-08-22

REPOSITORIES: Pride

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			Action	DRS
	OMG3964.sf3	Other
	OMG3964_A1.raw	Raw
	OMG3964_A2.raw	Raw
	OMG3964_A9.raw	Raw
	OMG3964_B1.raw	Raw

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The FAM114A proteins are adaptors for the recycling of Golgi enzymes

Project description:The Golgi apparatus contains many resident enzymes that must remain in place whilst their substrates flow through on their journey from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the rims of the Golgi stack to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and at least one COPI adaptor protein, GOLPH3, has been shown to recruit enzymes into vesicles in a specific part of the stack. We have used proximity biotinylation to identify further components of intra-Golgi transport vesicles and found FAM114A2, an uncharacterised cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1 showed that they bind to numerous Golgi resident proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not result in substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Generation of Drosophila mutants lacking FAM114A revealed defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins are COPI vesicle resident proteins that bind to Golgi enzymes and so are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

2024-08-22 | PXD054030 | Pride

The FAM114A proteins are adaptors for the recycling of Golgi enzymes - Fig4b-E

2024-08-22 | PXD054480 | Pride

The FAM114A proteins are adaptors for the recycling of Golgi enzymes - Figu 2SA

2024-08-22 | PXD054197 | Pride

Targeted Sphingolipid analysis of HeLa knockout for expression of GRASP55

Project description:Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis-trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 acts by binding to these enzymes and preventing their entry to COPI derived retrograde transport vesicles thus concentrating them in the trans-Golgi. In genome edited cells lacking GRASP55 the enzymes relocate to cis-Golgi. Here we evaluated the impact of deleting GRASP55 on sphingolipid composition of HeLa cells by targeted lipid analysis.

2021-07-18 | ST001877 | MetabolomicsWorkbench

Dissecting the secretory pathway with electrophoresis reveals intra-Golgi trafficking pathways and enables compilation of sub-Golgi and sub-ER proteomes.

Project description:The Golgi is the hub of the eukaryotic secretory pathway, trafficking proteins and lipids, as well as synthesizing complex sugars. Different biosynthetic reactions are associated with different compartments of its complex architecture. Although pre- and post-Golgi trafficking has been much studied, comparatively little is known about intra-Golgi organization. In this study, secretory vesicles and organelles were separated along an electrophoretic gradient at sub-Golgi resolution, presenting snapshots of the changing relative abundance of hundreds of resident and cargo proteins and glycans in transit through the ER, Golgi compartments and post-Golgi compartments. Furthermore, grouped features in migration profiles reveal the dominant intra-Golgi protein trafficking pathways, showing separate routes for cargo and different groups of resident proteins. As few structural characteristics of proteins or sequence motifs have been associated with specific regions of the Golgi stack, we also carried out a comparative analysis of the transmembrane regions of resident proteins associated with the main migratory profiles identifying the presence of charge amino acids adjacent to the transmembrane helix, exoplasmic Ser and Thr content and helix composition as likely contributors to protein sorting mechanisms.

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Alfa2-COP is involved in early secretory traffic in Arabidopsis and it is required for plant growth

Project description:The loss-of-function mutants of alfa1-COP (alfa1-COP-1) and alfa2-COP (alfa2-COP1), the two alfa-COP isoforms of Arabidopsis. Unlike in alfa1-COP mutant, the alfa2-COP mutant displays defects in plant growth, including small rosettes, stems and roots and mislocalization of p24a5, a protein containing a C-terminal dylisine motif involved in COPI binding. In addition, it exhibited abnormal morphology of the Golgi apparatus. Global expression analyses of the alfa2-COP mutant versus wild-type Arabidopsis plants reveal altered expression of plant cell wall-associated genes. A high expression of the COPII sec31A isoform was observed in the alfa2-COP mutant that also occurs in a mutant of an upstream gene of COPI assembly, the ARF-GEF GNL1

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Proteomic Profiling of Mammalian COPI Vesicles

Project description:By combining in vitro COPI vesicle reconstitutions and SILAC-based mass spectrometric analysis this study aims to identify the core proteome of mammalian COPI vesicles. Furthermore proteomes generated from vesicles with γ- and ζ-COP as well as Arf isoforms have been investigated with the same approach to identify isoform-dependent cargo proteins.

2022-02-23 | PXD012129 | Pride

Analysis of short chain phosphatidylcholine (PC) on the Golgi membrane

Project description:Studies on vesicle formation by the Coat Protein I (COPI) complex have contributed to a basic understanding of how vesicular transport is initiated. We have identified that short chain lipids promote membrane properties that are conducive for fission. Here we investigated short chain PCs on Golgi membrane. These findings will advance the understanding of how lipid geometry contributes to membrane deformation needed for vesicle fission.

2019-04-25 | ST001177 | MetabolomicsWorkbench

Shared and specific functions of Arfs 1-5 at the golgi revealed by systematic knockouts

Project description:DP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4, and Arf4+5 could not be deleted simultaneously. Class I Arfs are non-essential and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes.

2021-11-02 | PXD028846 | Pride

A WDR35-dependent coatomer transports ciliary membrane proteins from the Golgi to the cilia

Project description:Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo inside cilia. However, the mechanism of selective transport of cargo to cilia and entry across the diffusion barrier is not well understood. WDR35/IFT121 is a component of the IFT-A complex, best known for ciliary retrograde transport. Small Wdr35 mutant cilia are formed but fail to enrich in diverse classes of ciliary membrane proteins. Whilst the assembly of the IFT-B complex is unaffected, these exhibit retrograde trafficking defects in Wdr35 mutants. In contrast, the IFT-A peripheral components are degraded and core components remain stuck at the cilia base. The deep sequence homology and structural similarity of WDR35 and other IFT-As to the coatomer COPI proteins α and ß’, and accumulation of electron-light vesicles around Wdr35 mutant cilia, suggest that WDR35 is required to form coated vesicles delivering cargo from the Golgi necessary for cilia elongation. This is the first insitu proof towards a novel coatomer function for WDR35 and likely other IFT-A proteins in transporting ciliary membrane proteins from the Golgi.

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