Rab GTPases and tethering in the yeast endocytic pathway

Lachmann, Jens; Ungermann, Christian; Engelbrecht-Vandré, Siegfried

doi:10.4161/sgtp.2.3.16701

Cited by 27 publications

(25 citation statements)

References 43 publications

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“…In mammalian cells, the key proteins in this process are EEA1 (homolog of yeast Fab1), which binds to PI3P (34,42) and is thought to recruit and to effect Rab5 (homolog of yeast Vps21) (10). Rab5 in turn tethers vesicles (16,37) and interacts with the fusion complex CORVET (43) and through EEA1 with SNARE (44). However, owing to the lack of a direct measurement, it is still debated whether PI3P is produced on incoming vesicles, fused vesicles, or endosomes.…”

Section: Challenges For Counting Single Molecules With Superresolutionmentioning

confidence: 99%

“…PI3P is required for endocytosis (7) and membrane transport to early and late endosomes (8,9). PI3P regulates the recruitment of proteins (10), such as Rab GTPases, which coordinate many aspects of vesicle identity (11,12) and maturation to endosomes, including tethering and fusion (13)(14)(15)(16). These master regulators also modulate PI3P production; thus, there is mutual regulation between PI3P and the Rabs (17).…”

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confidence: 99%

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Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Puchner

Walter

Kasper

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

172

213

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Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.S ingle-cell analysis of protein abundance has revealed cell-tocell variation in the form of phenotypic (extrinsic) heterogeneity and intrinsic variation (1). Dynamic processes such as the cell cycle, endocytosis, and meiosis are regulated but also influenced by stochastic events involving small numbers of molecules (e.g., DNA transcription) (2, 3). Similarly, the size and molecular composition of small subcellular organelles are dynamically regulated but still subject to stochastic noise. Studying the biomolecular composition and size of organelles will illuminate the regulation and noise in these dynamically stable systems. A major challenge for such exploration, however, is the development of a combined approach for both resolving small structures below the optical diffraction limit and simultaneously counting biomolecules over several orders of magnitude (Fig. 1A).In the endocytic pathway, both vesicle morphology and biomolecular composition are dynamically regulated along a maturation path. Formation of vesicles, tethering, fusion, and maturation to endosomes are controlled by over 60 proteins in concert with conversion of phosphoinositides (PIs) (4, 5). Phosphatases and PI-kinases produce phosphatidylinositol 3-phosphate (PI3P) from plasma membrane phospholipids (6). PI3P is required for endocytosis (7) and membrane transport to early and late endosomes (8, 9). PI3P regulates the recruitment of proteins (10), such as Rab GTPases, which coordinate many aspects of vesicle identity (11, 12) and maturation to endosomes, including tethe...

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Section: Challenges For Counting Single Molecules With Superresolutionmentioning

confidence: 99%

mentioning

confidence: 99%

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Puchner

Walter

Kasper

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

172

213

View full text Add to dashboard Cite

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“…In addition, Rabs depend on a GTPase activating protein (GAP) to hydrolyze the GTP to GDP, and then become substrates of the GDI chaperone, which can extract the Rab-GDP and keep it soluble in the cytoplasm (Lachmann et al, 2011;Hutagalung and Novick 2011;Itzen and Goody 2011;Barr, 2013).…”

Section: Introductionmentioning

confidence: 99%

The Mon1-Ccz1 GEF activates the Rab7 GTPase Ypt7 via a longin fold-Rab interface and association with PI-3-P-positive membranes

Cabrera

Nordmann

Perz

et al. 2014

Journal of Cell Science

Self Cite

103

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To function in fusion and signaling, Rab GTPases need to be converted into their active GTP form. We previously identified the conserved Mon1-Ccz1 complex as the guanine nucleotide exchange factor (GEF) of the yeast Rab7 GTPase Ypt7. To address the possible GEF mechanism, we generated a homology model of the predicted longin domains of Mon1 and Ccz1 using the Rabbinding surface of the TRAPP complex as a template. On the basis of this, we identified mutations in both yeast Mon1 and Ccz1 that block Ypt7 activation, without affecting heterodimer formation and intracellular localization of Mon1 and Ccz1 at endosomes. Strikingly, the activity of the isolated Mon1-Ccz1 complex for Ypt7 is highly stimulated on membranes, and is promoted by the same anionic phospholipids such as phosphatidylinositol-3-phosphate (PI3P), which also support membrane association of the GEF complex. Our data imply that the GEF activity of the Mon1-Ccz1 complex towards Rab7/Ypt7 requires the interface formed by their longin domains and profits strongly from its association with the organelle surface.

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“…SNAREs fold in a zipper-like manner into tight four-helix complexes. This brings the two membranes into close contact and thus mediates bilayer mixing (1)(2)(3). The main regulation of membrane fusion appears to occur at the level of Rabs and tethering proteins.…”

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confidence: 99%

Guanine Nucleotide Exchange Factors (GEFs) Have a Critical but Not Exclusive Role in Organelle Localization of Rab GTPases

Cabrera

Ungermann

2013

Journal of Biological Chemistry

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Background: Rab localization has been ascribed to guanine nucleotide exchange factor (GEF) localization. Results: GEF deletions result in mislocalization of Rabs to other membranes, which can be bypassed by a Rab mutant. Conclusion: GEFs are critical for Rab localization, but Rabs also have a GEF-independent ability to localize correctly. Significance: Our data reveal that both GEFs and Rabs contribute to Rab localization in cells.

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Rab GTPases and tethering in the yeast endocytic pathway

Cited by 27 publications

References 43 publications

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

The Mon1-Ccz1 GEF activates the Rab7 GTPase Ypt7 via a longin fold-Rab interface and association with PI-3-P-positive membranes

Guanine Nucleotide Exchange Factors (GEFs) Have a Critical but Not Exclusive Role in Organelle Localization of Rab GTPases

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