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

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): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Catarina Franco

LAB HEAD: Sean Munro

PROVIDER: PXD054197 | Pride | 2024-08-22

REPOSITORIES: Pride

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			Action	DRS
	4994_SAMPLES.xlsx	Xlsx
	OMG4994_TMT_F1.raw	Raw
	OMG4994_TMT_F10.raw	Raw
	OMG4994_TMT_F11.raw	Raw
	OMG4994_TMT_F12.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

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

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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.

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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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Proteomic Profiling of Mammalian COPI Vesicles

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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.

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

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