Rab7 palmitoylation is required for efficient endosome-to-TGN trafficking

Modica, Graziana; Skorobogata, Olga; Sauvageau, Étienne; Vissa, Adriano; Yip, Christopher M.; Kim, Peter K.; Würtele, Hugo; Lefrançois, Stéphane

doi:10.1242/jcs.199729

Cited by 39 publications

(91 citation statements)

References 47 publications

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“…Some of the apical vesicular structures containing ntChx were also positive for Rab7 (Figure 2c), a protein involved in early-to-late endosome maturation, modulation of ER homeostasis and stress, and endosome-to-TGN trafficking. [23][24][25] Similar to EEA1, Rab7/ntChx co-localizations were restricted to the apical region of enterocytes, with large Rab7 + vesicles being present that were consistent with lysosomal structure but which did not contain ntChx.…”

Section: In Vivo A→b Transcytosis Across Rat Intestinal Epitheliummentioning

confidence: 88%

Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis

et al. 2020

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Cholix (Chx) is expressed by the intestinal pathogen Vibrio cholerae as a single chain of 634 amino acids (~70.7 kDa protein) that folds into three distinct domains, with elements of the second and third domains being involved in accessing the cytoplasm of nonpolarized cells and inciting cell death via ADP-ribosylation of elongation factor 2, respectively. In order to reach nonpolarized cells within the intestinal lamina propria, however, Chx must cross the polarized epithelial barrier in an intact form. Here, we provide in vitro and in vivo demonstrations that a nontoxic Chx transports across intestinal epithelium via a vesicular trafficking pathway that rapidly achieves vesicular apical to basal (A→B) transcytosis and avoids routing to lysosomes. Specifically, Chx traffics in apical endocytic Rab7 + vesicles and in basal exocytic Rab11 + vesicles with a transition between these domains occurring in the ER-Golgi intermediate compartment (ERGIC) through interactions with the lectin mannose-binding protein 1 (LMAN1) protein that undergoes an intracellular redistribution that coincides with the reorganization of COPI + and COPII + vesicular structures. Truncation studies demonstrated that domain I of Chx alone was sufficient to efficiently complete A→B transcytosis and capable of ferrying genetically conjoined human growth hormone (hGH). These studies provide evidence for a pathophysiological strategy where native Chx exotoxin secreted in the intestinal lumen by nonpandemic V. cholerae can reach nonpolarized cells within the lamina propria in an intact form by using a nondestructive pathway to cross in the intestinal epithelial that appears useful for oral delivery of biopharmaceuticals. One-Sentence Summary: Elements within the first domain of the Cholix exotoxin protein are essential and sufficient for the apical to basal transcytosis of this Vibrio cholerae-derived virulence factor across polarized intestinal epithelial cells.

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Section: In Vivo A→b Transcytosis Across Rat Intestinal Epitheliummentioning

confidence: 88%

Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis

et al. 2020

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“…Renilla Luciferase II (RlucII) was fused at the N-terminus to wild-type Rab7A (RlucII-Rab7A). As previously shown, this tag had little effect on the distribution or function of Rab7A as expressing RlucII-Rab7A in Rab7A knockout cells (Rab7A-KO) rescued retromer recruitment (Modica et al, 2017). The energy acceptor green fluorescence protein 10 (GFP10) was fused at the N-terminus of wild-type and various point mutation containing CLN3 (GFP10-CLN3, GFP10-CLN3 R334H , GFP10-CLN3 V330F , GFP10-CLN3 E295K , GFP10-CLN3 L101 P).…”

Section: Resultsmentioning

confidence: 83%

“…To determine if CLN3 is required for the membrane recruitment of Rab7A, we performed a membrane isolation experiment as we have previously done (Mamo et al, 2012; Modica et al, 2017), in wild-type, CLN3-KO and CLN3-KO HeLa cells expressing FLAG-CLN3 ( Figure 2a ). Our membrane separation was successful, as the integral membrane protein Lamp2 was found in the pellet fraction (P), while the cytosolic protein tubulin was found in the supernatant fraction (S) ( Figure 2a ).…”

Section: Resultsmentioning

confidence: 99%

“…However, in Rab7A depleted cells, CI-MPR was not efficiently recycled to the TGN, but was not degraded (Rojas et al, 2008). We repeated the membrane assay as above, but we included Rab7A-KO HeLa cells a as control, as we previously demonstrated a reduction of retromer recruitment in Rab7A-KO HEK293 cells (Modica et al, 2017). Lamp2 and tubulin were used as markers of membrane (P) and cytosolic fractions (S) respectively ( Figure 2c ).…”

Section: Resultsmentioning

confidence: 99%

“…Compared to wild-type HeLa cells, we found a 2 fold increase in the BRET 50 values for Rab7A binding to Vps26A in CLN3-KO cells, suggesting a weaker interaction ( Figure 3b ). We previously demonstrated that the retromer/Rab7A interaction requires the palmitoylation of Rab7A (Modica et al, 2017). We tested whether or not the palmitoylation level of Rab7A was lower in CLN3-KO versus wild-type HeLa cells using Acyl-RAC, a technique to determine the palmitoylation status of a protein ( Figure 3c ).…”

Section: Resultsmentioning

confidence: 99%

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CLN3 regulates endosomal function by modulating Rab7A activity

Yasa

Modica

Sauvageau

et al. 2019

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Mutations in CLN3 are a cause of juvenile NCL (JNCL), also known as Batten Disease.Clinical manifestations includes cognitive regression, progressive loss of vision and motor function, epileptic seizures, and a significantly reduced lifespan. CLN3 localizes to endosomes and lysosomes, and has been implicated in intracellular trafficking and autophagy. However, the precise molecular function of CLN3 remains to be elucidated. We show that CLN3 interacts with Rab7A, a small GTPase that regulates several functions at late endosomes. We found that CLN3 is required for the efficient endosome-to-TGN trafficking of the lysosomal sorting receptors by regulating the Rab7A/retromer interaction. In cells lacking CLN3 or expressing CLN3 harbouring a disease-causing mutation, the lysosomal sorting receptors were degraded.We also demonstrated that CLN3 is required for the Rab7A/PLEKHM1 interaction, which is required for autophagosome/lysosome fusion. Overall, our data provides a molecular explanation behind phenotypes observed in JNCL.

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The functional roles of retromer in Parkinson's disease

2017

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The endosomal system is critical for the maintenance of intracellular homeostasis, and defects in this system are often linked to neurological disorders. The retromer complex is a critical coordinator of endosomal dynamics and has functional roles in multiple cellular processes through sorting cargoes from endosomes to the trans-Golgi network (TGN) or to the plasma membrane. Mammalian retromer comprises a core Vps26-Vps35-Vps29 trimer and associates with a range of proteins to generate endosomal tubular-vesicular carriers. Alterations in retromer function or its molecular organization have been a rising risk factor for Parkinson's disease (PD). Although genetic evidence has shown several variants within retromer in late-onset PD cases, the exact molecular machineries by which retromer variants induce the development of PD are still not completely elucidated. In this Review, we will focus on the functional roles of retromer in neuronal health, summarize advances in defining the cellular pathological phenotype caused by retromer deficiency or PD-linked retromer variants and discuss the potential clues of how retromer deregulation may contribute to PD pathogenesis.Keywords: autophagy; endosomal trafficking; lysosome; mitochondria; Parkinson's disease (PD); retromer The endosomal network is a highly dynamic and orchestrated system, which serves a vital function in coordinating the communication and exchange of associated proteins and lipids between intracellular membrane-bounded compartments. Once within the network, cargo molecules originating from the plasma membrane and biosynthetic pathways are targeted to a common sorting station, the early endosome, from where cargoes are sorted towards specialized intracellular locations following multiple trafficking routes. They can be either sorted into late endosomes/ lysosomes for degradation or retrieved to the plasma membrane or the trans-Golgi network (TGN). The efficient and accurate delivery of cargo contents within intracellular compartments is critical for the maintenance of intracellular homeostasis, and defects on it are often associated with multiple human diseases. The evolutionary conserved protein complex, termed Abbreviations AMPAR, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor; APEX, ascorbate peroxidase; APP, b-amyloid precursor protein; ATG9A, autophagy-associated protein; Ab, elevated b-amyloid peptide; BAR, Bin-Amphiphysin-Rvs; BDNF, brain-derived neurotrophic factor; CI-M6PR, cation-independent mannose 6-phosphate receptor; CLN5, ceroid lipouscinosis neuronal protein-5; CMA, chaperon-mediated autophagy; Crb, crumb; DA, dopaminergic; DMTI-II, divalent metal transporter II; DRD1, dopamine receptor D1; Drp1, dynamin-related protein 1; EHD1, Eps15 homology domain-containing protein-1; FERM, 4.1/ezrin/radixin/moesin; GLUT1, glucose transporter 1; Kir3, G protein-gated inward rectifying potassium channels; Lamp2a, lysosome-associated membrane glycoprotein 2a; LBs, Lewy bodies; MAPL, mitochondria-anchored protein ligase; MDVs, mitochondria-de...

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Rab7 palmitoylation is required for efficient endosome-to-TGN trafficking

Cited by 39 publications

References 47 publications

Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis

Cholix protein domain I functions as a carrier element for efficient apical to basal epithelial transcytosis

CLN3 regulates endosomal function by modulating Rab7A activity

The functional roles of retromer in Parkinson's disease

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