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The Rab family of small guanosine triphosphatases (GTPases) are nucleotide-dependent switches. Mutations in Rabs can result in human diseases. Rab7a and Rab7b transition from early endosomes to lysosomes and are presumed to function similarly. Most studies look at Rab7a, less on Rab7b, with the underlying assumption they function similarly. There have yet to be articles comparing them side by side. Whilst cloning Rab7 homologues, we identified splice isoforms for Rab7b only. These splice isoforms, Rab7b2 and Rab7bx8 lacking different exons, have not been previously characterized but suggest alternative function(s) for Rab7b. Thus, we hypothesize that Rab7 homologues have distinct functions. Here, we compare Rab7a and Rab7b nucleotide mutants locked in GDP-bound (Rab7T22N), GTP-bound (Rab7Q67L), nucleotide-free (Rab7aN125I/Rab7bN124I) states and characterized localization of the Rab7b splice isoforms. HeLa cells were transiently transfected with fluorescently tagged Rab7 reporters. Confocal images were processed with ImageJ and analyzed with SPSS. Rab7a and Rab7b nucleotide mutants were significantly different to one another. Approximately 50% of Rab7b splice isoform-expressing cells had aggregated vesicles, which were phenotypically different from Rab7b vesicles. Rab7a and Rab7b vesicles shared approximately 60% colocalization with each other, while Rab7b vesicles preferentially localized to the Trans Golgi Network. Our results suggest Rab7b is distinct from Rab7a, and Rab7b splice isoforms have different biological functions.

Rab7 is a GTPase that controls late endosome and lysosome trafficking. Recent studies have demonstrated that Rab7 is ubiquitinated, a post-translational modification mediated by an enzymatic cascade. To date, only one ubiquitin E3 ligase and one deubiquitinase have been identified in regulating Rab7 ubiquitination. Here, we report that RNF167, a transmembrane endolysosomal ubiquitin ligase, can ubiquitinate Rab7. Using immunoprecipitation and in vitro ubiquitination assays, we demonstrate that Rab7 is a direct substrate of RNF167. Subcellular fractionation indicates that RNF167 activity maintains Rab7′s membrane localization. Epifluorescence microscopy in HeLa cells shows that Rab7-positive vesicles are larger under conditions enabling Rab7 ubiquitination by RNF167. Characterization of its ubiquitination reveals that Rab7 must be in its GTP-bound active form for membrane anchoring and, thus, accessible for RNF167-mediated ubiquitin attachment. Cellular distribution analyses of lysosome marker Lamp1 show that vesicle positioning is independent of Rab7 and RNF167 expression and that Rab7 endosomal localization is not affected by RNF167 knockdown. However, both Rab7 and RNF167 depletion affect each other’s lysosomal localization. Finally, this study demonstrates that the RNF167-mediated ubiquitination of Rab7 GTPase is impaired by variants of Charcot–Marie–Tooth Type 2B disease. This study identified RNF167 as a new ubiquitin ligase for Rab7 while expanding our knowledge of the mechanisms underlying the ubiquitination of Rab7.

Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson’s disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.

Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate–binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.

Damaged mitochondria can be selectively eliminated by mitophagy. Although two gene products mutated in Parkinson’s disease, PINK1, and Parkin have been found to play a central role in triggering mitophagy in mammals, how the pre-autophagosomal isolation membrane selectively and accurately engulfs damaged mitochondria remains unclear. In this study, we demonstrate that TBC1D15, a mitochondrial Rab GTPase-activating protein (Rab-GAP), governs autophagosome biogenesis and morphology downstream of Parkin activation. To constrain autophagosome morphogenesis to that of the cargo, TBC1D15 inhibits Rab7 activity and associates with both the mitochondria through binding Fis1 and the isolation membrane through the interactions with LC3/GABARAP family members. Another TBC family member TBC1D17, also participates in mitophagy and forms homodimers and heterodimers with TBC1D15. These results demonstrate that TBC1D15 and TBC1D17 mediate proper autophagic encapsulation of mitochondria by regulating Rab7 activity at the interface between mitochondria and isolation membranes. Parkinson disease is a common degenerative brain disorder that causes tremors, muscle stiffening, and slowing down of movement. Scientists believe that these symptoms are caused by a progressive loss of brain cells called dopaminergic neurons, which help regulate movement. Most cases have no obvious genetic cause, but around 15% of people with the disease have a close relative who also has the disease, and mutations in the genes encoding two proteins—PINK1 and Parkin—have been identified as prime suspects in familial Parkinson disease. These proteins help to eliminate damaged mitochondria from cells. In addition to producing the energy that cells need to function, mitochondria also help to trigger cell death. Pesticides and other chemicals linked to non-familial cases of Parkinson disease also damage mitochondria. Taken together, this evidence suggests that the accumulation of damaged mitochondria may contribute to the excessive loss of dopaminergic neurons that is seen in both forms of the disease. Yamano et al. provide new details on the ways that autophagosomes—structures that help cells to recycle nutrients and remove debris—destroy mitochondria. Previous studies have shown that when a mitochondrion is damaged, PINK1 sends a signal to Parkin, which then helps to recruit the proteins that are needed to form an autophagosome around the damaged mitochondrion. However, the identity of the proteins that guide the formation of the autophagosome remained a mystery. Yamano et al. have now identified two of these proteins and helped to explain their specific roles in the assembly of autophagosomes. The two proteins, which are called TBC1D15 and TBC1D17, are both GAP proteins, which are well known for their role in deactivating enzymes called RAB GTPases. Yamano et al. show that TBC1D15 binds to the damaged mitochondrion and also to the autophagosome as it grows around the mitochondrion. TBC1D15 also inhibits the action of an enzyme called Rab7 to prevent excessive growth of the autophagosome. TBC1D17 has a similar role. The work of Yamano et al. indicates that Parkin activates Rab7, perhaps by placing chains of a protein called ubiquitin on mitochondria, which would mean that an unexpected new step in this pathway remains to be discovered. Understanding how Parkin activates Rab7 could help identify new targets for drugs that might treat Parkinson disease.

Background Neuroinflammation and oxidative stress plays an important role in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). This study is the first to show that activation of autophagy protein nuclear receptor binding factor 2 (NRBF2) could reduce endoplasmic reticulum stress (ERS)-associated inflammation and oxidative stress after SAH. Methods Male C57BL/6J mice were subjected to endovascular perforation to establish a model of SAH. NRBF2 overexpression adeno-associated virus (AAV), NRBF2 small interfering RNAs (siRNA), lysosomal inhibitor-chloroquine (CQ), and late endosome GTPase Rab7 receptor antagonist-CID1067700 (CID) were used to investigate the role of NRBF2 in EBI after SAH. Neurological tests, brain water content, western blotting and immunofluorescence staining were evaluated. Results Our study found that the level of NRBF2 was increased after SAH and peaked at 24 h after SAH. In addition, we found that the overexpression of NRBF2 significantly improved neurobehavioral scores and reduced ERS, oxidative stress, and neuroinflammation in SAH, whereas the inhibition of NRBF2 exacerbated these phenotypes. In terms of mechanism, NRBF2 overexpression significantly promoted autophagosome maturation, with the downregulation of CHOP, Romo-1, TXNIP, NLRP3, TNF-α, and IL-1β expression through interaction with Rab7. The protective effect of NRBF2 on ERS-associated neuroinflammation and oxidative stress after SAH was eliminated by treatment with CQ. Meanwhile, it was also reversed by intraperitoneal injection of CID. Moreover, the MIT domain of NRBF2 was identified as a critical binding site that interacts with Rab7 and thereby promotes autophagosome maturation. Conclusion Our data provide evidence that the autophagy protein NRBF2 has a protective effect on endoplasmic reticulum stress-associated neuroinflammation and oxidative stress by promoting autophagosome maturation through interactions with Rab7 after SAH.

Rab7 is a small GTPase that belongs to the Rab family and controls transport to late endocytic compartments such as late endosomes and lysosomes. The mechanism of action of Rab7 in the late endocytic pathway has been extensively studied. Rab7 is fundamental for lysosomal biogenesis, positioning and functions, and for trafficking and degradation of several signaling receptors, thus also having implications on signal transduction. Several Rab7 interacting proteins have being identified leading to the discovery of a number of different important functions, beside its established role in endocytosis. Furthermore, Rab7 has specific functions in neurons. This review highlights and discusses the role and the importance of Rab7 on different cellular pathways and processes.

Background Cotton serves as a primary source of natural fibers crucial for the textile industry. However, environmental elements such as drought have posed challenges to cotton cultivation, resulting in adverse impacts on both production and fiber quality. Improving cotton’s resilience to drought could mitigate yield losses and foster the expansion of cotton farming. Rab7 protein, widely present in organisms, controls the degradation and recycling of cargo, and has a potential role in biotic and abiotic tolerance. However, comprehensive exploration of the Rab7 gene family in Gossypium remains scarce. Results Herein, we identified a total of 10, 10, 20, and 20 Rab7 genes through genome-wide analysis in Gossypium arboreum , Gossypium raimondii , Gossypium hirsutum , and Gossypium barbadense , respectively. Collinearity analysis unveiled the pivotal role of whole genome or segmental duplication events in the expansion of GhRab7s . Study of gene architecture, conserved protein motifs, and domains suggested the conservation of structure and function throughout evolution. Exploration of cis-regulatory elements revealed the responsiveness of GhRab7 genes to abiotic stress, corroborated by transcriptome analysis under diverse environmental stresses. Notably, the greatly induced expression of GhRab7B3-A under drought treatment prompted us to investigate its function through virus-induced gene silencing (VIGS) assays. Silencing GhRab7B3-A led to exacerbated dehydration and wilting compared with the control. Additionally, inhibition of stomatal closure, antioxidant enzyme activities and expression patterns of genes responsive to abiotic stress were observed in GhRab7B3-A silenced plants. Conclusions This study sheds light on Rab7 members in cotton, identifies a gene linked to drought stress, and paves the way for additional investigation of Rab7 genes associated with drought stress tolerance.

Cardiomyocyte apoptosis critically contributes to ischemic heart failure (IHF) progression. While the endosome-lysosome system governs cellular homeostasis, the functional significance of its master regulator RAB7 in cardiac pathophysiology remains unexplored. Using myocardial infarction (MI) models via left anterior descending coronary artery ligation in cardiomyocyte-specific RAB7 knockout mice and adeno-associated virus-mediated RAB7 overexpression models, we assessed cardiac function and adverse remodeling through echocardiography and pathophysiological assessment. Mitophagy flux was quantified using mt-Keima mice and confocal imaging. Molecular mechanisms were dissected through immunoprecipitation coupled with mass spectrometry (IP-MS) analysis and molecular experiment validation. RAB7 expression decreased in ischemic myocardium. Cardiomyocyte-specific RAB7 ablation exacerbated while RAB7 overexpression attenuated post-MI cardiac dysfunction and maladaptive remodeling. RAB7 enhanced mitophagic clearance of damaged mitochondria, reducing cardiomyocyte apoptosis under ischemic stress both and . Mechanistically, TUFM, a mitochondrial translation elongation factor, was identified as a novel effector of RAB7. RAB7 facilitated the recruitment of TUFM and LC3 to damaged mitochondria, thereby enhancing mitophagy. TUFM knockdown significantly diminished the protective effects of RAB7 on mitophagy and cardiomyocyte survival. Finally, administration of ML-098, a chemical RAB7 activator, promoted mitophagy and mitigated IHF progression in mice. We identify RAB7 as a novel coordinator of cardioprotective mitophagy through TUFM-mediated machinery assembly. The RAB7-TUFM axis represents a therapeutic target for IHF that warrants further clinical evaluation.