Analysis of Rab GTPases

Langemeyer, Lars; Barr, Francis A.

doi:10.1002/0471143030.cb1518s57

Cited by 6 publications

(7 citation statements)

References 13 publications

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“…This quality can indicate whether a Rab interactor is, for instance, an effector. Effectors are expected to localize to membranes where active Rabs are found and should interact preferentially with GTP-bound Rabs 33 . We had noticed previously that Drp1 indeed is found on light ER membranes like active Rab32 22 .…”

Section: Resultsmentioning

confidence: 95%

Interaction with the effector dynamin-related protein 1 (Drp1) is an ancient function of Rab32 subfamily proteins

et al. 2014

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The mitochondria-associated membrane (MAM) is an endoplasmic reticulum (ER) domain that forms contacts with mitochondria and accommodates Ca2+ transfer between the two organelles. The GTPase Rab32 regulates this function of the MAM via determining the localization of the Ca2+ regulatory transmembrane protein calnexin to the MAM. Another function of the MAM is the regulation of mitochondrial dynamics mediated by GTPases such as dynamin-related protein 1 (Drp1). Consistent with the importance of the MAM for mitochondrial dynamics and the role of Rab32 in MAM enrichment, the inactivation of Rab32 leads to mitochondrial collapse around the nucleus. However, Rab32 and related Rabs also perform intracellular functions at locations other than the MAM including melanosomal trafficking, autophagosome formation and maturation, and retrograde trafficking to the trans-Golgi network (TGN). This plethora of functions raises questions concerning the original cellular role of Rab32 in the last common ancestor of animals and its possible role in the last eukaryotic common ancestor (LECA). Our results now shed light on this conundrum and identify a role in Drp1-mediated mitochondrial dynamics as one common denominator of this group of Rabs, which includes the paralogues Rab32A and Rab32B, as well as the more recently derived Rab29 and Rab38 proteins. Moreover, we provide evidence that this mitochondrial function is dictated by the extent of ER-association of Rab32 family proteins.

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Section: Resultsmentioning

confidence: 95%

Interaction with the effector dynamin-related protein 1 (Drp1) is an ancient function of Rab32 subfamily proteins

et al. 2014

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“…31,32 Similar regulatory mechanisms have also been reported for Rabs in higher eukaryotes. 33,34 Other studies show that the hypervariable C-terminal domain of each Rab is instructive for insertion into specific membranes, 35 and that active Rabs are able to induce self-amplifying loops by generating functional lipid micro-domains. 16,36 Interestingly, on the same membrane entity multiple different Rab micro-domains can coexist.…”

Section: The Rab Activity Cycle: Hard Facts and Open Questionsmentioning

confidence: 99%

Rabs on the fly: Functions of Rab GTPases during development

et al. 2017

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The organization of intracellular transport processes is adapted specifically to different cell types, developmental stages, and physiologic requirements. Some protein traffic routes are universal to all cells and constitutively active, while other routes are cell-type specific, transient, and induced under particular conditions only. Small GTPases of the Rab (Ras related in brain) subfamily are conserved across eukaryotes and regulate most intracellular transit pathways. The complete sets of Rab proteins have been identified in model organisms, and molecular principles underlying Rab functions have been uncovered. Rabs provide intracellular landmarks that define intracellular transport sequences. Nevertheless, it remains a challenge to systematically map the subcellular distribution of all Rabs and their functional interrelations. This task requires novel tools to precisely describe and manipulate the Rab machinery in vivo. Here we discuss recent findings about Rab roles during development and we consider novel approaches to investigate Rab functions in vivo.

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“…We have used this end-point assay in a number of previous studies to directly compare the GEF activity of a candidate regulatory factor against a panel of different GTPases under the same experimental conditions 27 29 . The purification of the GTPases and the assay itself have been described in detail 30 . The well-characterized GEF Rabex5, and its substrate, the GTPase Rab5a 31 , provided a positive control for GEF activity.…”

Section: Resultsmentioning

confidence: 99%

“…For TD-60 GEF activity, we performed a GDP-release assay as previously described 30 70 . In brief, 10 μg of each pre-selected GST-tagged GTPase was added to the following mixture (125 μM EDTA, 50 mM HEPES-NaOH pH 6.8, 10 μM Mg-GDP, 0.1 mg ml −1 bovine serum albumin), plus 5 μCi [3H]-GDP (10 mCi ml −1 ; 5,000 Ci mmol −1 ) in a total volume of 200 μl.…”

Section: Methodsmentioning

confidence: 99%

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TD-60 links RalA GTPase function to the CPC in mitosis

et al. 2015

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TD-60 (also known as RCC2) is a highly conserved protein that structurally resembles the Ran guanine exchange factor (GEF) RCC1, but has not previously been shown to have GEF activity. TD-60 has a typical chromosomal passenger complex (CPC) distribution in mitotic cells, but associates with integrin complexes and is involved in cell motility during interphase. Here we show that TD-60 exhibits GEF activity, in vitro and in cells, for the small GTPase RalA. TD-60 or RalA depletion causes spindle abnormalities in prometaphase associated with abnormal centromeric accumulation of CPC components. TD-60 and RalA apparently work together to contribute to the regulation of kinetochore–microtubule interactions in early mitosis. Importantly, several mitotic phenotypes caused by TD-60 depletion are reverted by the expression of a GTP-locked mutant, RalA (Q72L). The demonstration that a small GTPase participates in the regulation of the CPC reveals a level of mitotic regulation not suspected in previous studies.

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Analysis of Rab GTPases

Cited by 6 publications

References 13 publications

Interaction with the effector dynamin-related protein 1 (Drp1) is an ancient function of Rab32 subfamily proteins

Interaction with the effector dynamin-related protein 1 (Drp1) is an ancient function of Rab32 subfamily proteins

Rabs on the fly: Functions of Rab GTPases during development

TD-60 links RalA GTPase function to the CPC in mitosis

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