Limits...
Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro.

Tamura N, Mima J - Biol Open (2014)

Bottom Line: Strikingly, we found that three specific human Rabs (endoplasmic reticulum/Golgi Rab2a, early endosomal Rab5a, and late endosomal/lysosomal Rab7a) strongly accelerated membrane aggregation of synthetic liposomes even in the absence of any additional components, such as classical tethers, tethering factors, and Rab effectors.This Rab-induced membrane aggregation was a reversible membrane tethering reaction that can be strictly controlled by the membrane recruitment of Rab proteins on both apposing membranes.Thus, our current reconstitution studies establish that membrane-anchored human Rab GTPases are an essential tethering factor to directly mediate membrane tethering events.

View Article: PubMed Central - PubMed

Affiliation: Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.

No MeSH data available.


Related in: MedlinePlus

CD spectra of purified human Rab GTPases.Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl2 (5 mM), and DTT (1 mM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4232769&req=5

f02: CD spectra of purified human Rab GTPases.Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl2 (5 mM), and DTT (1 mM).

Mentions: Rab GTPases are typically post-translationally modified by an isoprenyl lipid group at their C-terminal cysteine residues, which is required for membrane association of Rabs (Hutagalung and Novick, 2011). To mimic the membrane-bound state of native Rabs bearing the lipid anchor, the seven selected human Rabs were purified as the C-terminal polyhistidine-tagged forms (Rab-His12 proteins) that can be attached to liposome membranes bearing a DOGS-NTA lipid (1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]-succinyl}) (Fig. 1A, lanes 1–7). For a negative control, we also purified the His12-tagged form of human HRas, which is a similar Ras-family GTPase with a C-terminal lipid anchor but not functionally related to membrane tethering events (Fig. 1A, lane 8). All the purified Rab-His12 and HRas-His12 proteins retained their intrinsic GTP-hydrolysis activities, specifically converting GTP to GDP and a free phosphate group (Fig. 1B,C). In addition, we further characterized the purified Rab proteins by circular dichroism (CD) spectroscopy (Fig. 2). All the six Rab-His12 proteins tested, except Rab2a-His12, had similar far-UV CD spectra (Fig. 2) and comparable secondary structure contents which were estimated from the CD spectra using a K2D3 program (Table 1) (Louis-Jeune et al., 2012). These biochemical properties support that those six Rab-His12 proteins are a well-folded protein that indeed has the capacity to bind and hydrolyze a guanine nucleotide. However, as Rab2a-His12 showed significant differences from the other Rab-His12 proteins in the CD spectra and predicted secondary structure contents, it should be noted that our preparation of Rab2a-His12 likely contained some denatured or partially-denatured proteins.


Membrane-anchored human Rab GTPases directly mediate membrane tethering in vitro.

Tamura N, Mima J - Biol Open (2014)

CD spectra of purified human Rab GTPases.Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl2 (5 mM), and DTT (1 mM).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232769&req=5

f02: CD spectra of purified human Rab GTPases.Far-UV CD spectra of Rab1a-His12 (black), Rab2a-His12 (red), Rab3a-His12 (green), Rab4a-His12 (yellow), Rab5a-His12 (blue), Rab6a-His12 (pink), Rab7a-His12 (cyan), HRas-His12 (brown), untagged Rab5a (blue dashed line), and untagged Rab7a (cyan dashed line), in HN150 (20 mM Hepes-NaOH, pH 7.4, 150 mM NaCl) containing glycerol (10%), MgCl2 (5 mM), and DTT (1 mM).
Mentions: Rab GTPases are typically post-translationally modified by an isoprenyl lipid group at their C-terminal cysteine residues, which is required for membrane association of Rabs (Hutagalung and Novick, 2011). To mimic the membrane-bound state of native Rabs bearing the lipid anchor, the seven selected human Rabs were purified as the C-terminal polyhistidine-tagged forms (Rab-His12 proteins) that can be attached to liposome membranes bearing a DOGS-NTA lipid (1,2-dioleoyl-sn-glycero-3-{[N-(5-amino-1-carboxypentyl)iminodiacetic acid]-succinyl}) (Fig. 1A, lanes 1–7). For a negative control, we also purified the His12-tagged form of human HRas, which is a similar Ras-family GTPase with a C-terminal lipid anchor but not functionally related to membrane tethering events (Fig. 1A, lane 8). All the purified Rab-His12 and HRas-His12 proteins retained their intrinsic GTP-hydrolysis activities, specifically converting GTP to GDP and a free phosphate group (Fig. 1B,C). In addition, we further characterized the purified Rab proteins by circular dichroism (CD) spectroscopy (Fig. 2). All the six Rab-His12 proteins tested, except Rab2a-His12, had similar far-UV CD spectra (Fig. 2) and comparable secondary structure contents which were estimated from the CD spectra using a K2D3 program (Table 1) (Louis-Jeune et al., 2012). These biochemical properties support that those six Rab-His12 proteins are a well-folded protein that indeed has the capacity to bind and hydrolyze a guanine nucleotide. However, as Rab2a-His12 showed significant differences from the other Rab-His12 proteins in the CD spectra and predicted secondary structure contents, it should be noted that our preparation of Rab2a-His12 likely contained some denatured or partially-denatured proteins.

Bottom Line: Strikingly, we found that three specific human Rabs (endoplasmic reticulum/Golgi Rab2a, early endosomal Rab5a, and late endosomal/lysosomal Rab7a) strongly accelerated membrane aggregation of synthetic liposomes even in the absence of any additional components, such as classical tethers, tethering factors, and Rab effectors.This Rab-induced membrane aggregation was a reversible membrane tethering reaction that can be strictly controlled by the membrane recruitment of Rab proteins on both apposing membranes.Thus, our current reconstitution studies establish that membrane-anchored human Rab GTPases are an essential tethering factor to directly mediate membrane tethering events.

View Article: PubMed Central - PubMed

Affiliation: Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.

No MeSH data available.


Related in: MedlinePlus