The Arf-GAP Age2 localizes to the late-Golgi via a conserved amphipathic helix

Manzer, Kaitlyn M.; Fromme, J. Christopher

doi:10.1091/mbc.e23-07-0283

Cited by 5 publications

(2 citation statements)

References 100 publications

(157 reference statements)

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“…Amphipathic helices are common membrane-binding elements used by regulatory proteins that function at the Golgi. Several Arf and Rab GEFs and GAPs are known to use amphipathic helices to bind to the Golgi membrane 36,[57][58][59][60] . Our data suggest that the Rgp1 amphipathic helix is involved in Golgi membrane binding, yet it remains unclear how the Ric1-Rgp1 complex is recruited specifically to the surface of the Golgi, rather than another organelle.…”

Section: Discussionmentioning

confidence: 99%

Structural basis for Rab6 activation by the Ric1-Rgp1 complex

Feathers,

Vignogna,

Fromme

2024

Preprint

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Rab GTPases act as molecular switches to regulate organelle homeostasis and membrane trafficking. Rab6 plays a central role in regulating cargo flux through the Golgi and is activated via nucleotide exchange by the Ric1-Rgp1 protein complex. Ric1-Rgp1 is conserved throughout eukaryotes but the structural and mechanistic basis for its function has not been established. Here we report the cryoEM structure of a Ric1-Rgp1-Rab6 complex representing a key intermediate of the nucleotide exchange reaction. This structure reveals the overall architecture of the complex and enabled us to identify interactions critical for proper recognition and activation of Rab6 on the Golgi membrane surface. Ric1-Rgp1 interacts with the nucleotide-binding domain of Rab6 using an uncharacterized helical domain, which we establish as a novel RabGEF domain by identifying residues required for Rab6 nucleotide exchange. Unexpectedly, the complex uses an arrestin fold to interact with the Rab6 hypervariable domain, indicating that interactions with the unstructured C-terminal regions of Rab GTPases may be a common specificity mechanism used by their activators. Collectively, our findings provide a detailed mechanistic understanding of regulated Rab6 activation at the Golgi.

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Section: Discussionmentioning

confidence: 99%

Structural basis for Rab6 activation by the Ric1-Rgp1 complex

Feathers,

Vignogna,

Fromme

2024

Preprint

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“…We next sought to determine the relative importance of the D-loop and HDS2-GEF domain autoinhibitory interactions in vivo. We tested Sec7 mutants in a strain that was otherwise wild-type, as well as in a sensitized strain lacking Age2, a TGN-localized Arf-GAP (34,35). We reasoned that decreased inactivation of Arf1 due to loss of Age2 might exacerbate the effects of excessive Arf1 activation that may arise from loss of Sec7 autoinhibition.…”

Section: Alphafold Predicts An Active Conformation Of Sec7mentioning

confidence: 99%

Sec7 regulatory domains scaffold autoinhibited and active conformations

Brownfield,

Richardson,

Halaby

et al. 2023

Preprint

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The late stages of Golgi maturation involve a series of sequential trafficking events in which cargo-laden vesicles are produced and targeted to multiple distinct subcellular destinations. Each of these vesicle biogenesis events requires activation of an Arf GTPase by the Sec7/BIG guanine nucleotide exchange factor (GEF). Sec7 localization and activity is regulated by autoinhibition, positive feedback, and interaction with other GTPases. Although these mechanisms have been characterized biochemically, we lack a clear picture of how GEF localization and activity is modulated by these signals. Here we report the cryoEM structure of full-length Sec7 in its autoinhibited form, revealing the architecture of its multiple regulatory domains. We use functional experiments to determine the basis for autoinhibition and use structural predictions to produce a model for an active conformation of the GEF that is supported empirically. This study therefore elucidates the conformational transition that Sec7 undergoes to become active on the organelle membrane surface.

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Regulation of yeast polarized exocytosis by phosphoinositide lipids

Volpiana,

Nenadic,

Beh

2024

Cell. Mol. Life Sci.

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Phosphoinositides help steer membrane trafficking routes within eukaryotic cells. In polarized exocytosis, which targets vesicular cargo to sites of polarized growth at the plasma membrane (PM), the two phosphoinositides phosphatidylinositol 4-phosphate (PI4P) and its derivative phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) pave the pathway for vesicle transport from the Golgi to the PM. PI4P is a critical regulator of mechanisms that shape late Golgi membranes for vesicle biogenesis and release. Although enriched in vesicle membranes, PI4P is inexplicably removed from post-Golgi vesicles during their transit to the PM, which drives subsequent steps in exocytosis. At the PM, PI(4,5)P2 recruits effectors that establish polarized membrane sites for targeting the vesicular delivery of secretory cargo. The budding yeast Saccharomyces cerevisiae provides an elegant model to unravel the complexities of phosphoinositide regulation during polarized exocytosis. Here, we review how PI4P and PI(4,5)P2 promote yeast vesicle biogenesis, exocyst complex assembly and vesicle docking at polarized cortical sites, and suggest how these steps might impact related mechanisms of human disease.

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The Arf-GAP Age2 localizes to the late-Golgi via a conserved amphipathic helix

Cited by 5 publications

References 100 publications

Structural basis for Rab6 activation by the Ric1-Rgp1 complex

Structural basis for Rab6 activation by the Ric1-Rgp1 complex

Sec7 regulatory domains scaffold autoinhibited and active conformations

Regulation of yeast polarized exocytosis by phosphoinositide lipids

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