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GTRAP3-18 serves as a negative regulator of Rab1 in protein transport and neuronal differentiation.

Maier S, Reiterer V, Ruggiero AM, Rothstein JD, Thomas S, Dahm R, Sitte HH, Farhan H - J. Cell. Mol. Med. (2008)

Bottom Line: The effects on the early secretory pathway in HEK293 cells were: reduction of the rate of ER-to-Golgi transport of the vesicular stomatitis virus glycoprotein (VSVG), slowed accumulation of a Golgi marker plasmid in pre-Golgi structures after Brefeldin A treatment and inhibition of cargo concentration of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) into transpor complexes in HEK293 cells, an effect that could be completely reversed in the presence of an excess of Rab1.The inhibitory effect of GTRAP3-18 on neurite growth was rescued by co-expression with Rab1, supporting the conclusion that GTRAP 3-18 acted by inhibiting Rab1 action.Finally, we hypothesized that expression of GTRAP3-18 in the brain shoul be lower at stages of active synaptogenesis compared to early developmental stages.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology, Center for Biomolecular Medicine and Pharmacology, Medical University Vienna, Waehringer Strasse, Vienna, Austria.

ABSTRACT
Glutamate transporter associated protein 3-18 (GTRAP3-18) is an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family interacting with small Rab GTPases, which regulate intracellular trafficking events. Its impact on secretory trafficking has not been investigated. We report here that GTRAP3-18 has an inhibitory effect on Rab1, which is involved in ER-to-Golg trafficking. The effects on the early secretory pathway in HEK293 cells were: reduction of the rate of ER-to-Golgi transport of the vesicular stomatitis virus glycoprotein (VSVG), slowed accumulation of a Golgi marker plasmid in pre-Golgi structures after Brefeldin A treatment and inhibition of cargo concentration of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) into transpor complexes in HEK293 cells, an effect that could be completely reversed in the presence of an excess of Rab1. In accordance with the known role of Rab1 in neurite formation, overexpression of GTRAP3-18 significantly inhibited the length of outgrowing neurites in differentiated CAD cells. The inhibitory effect of GTRAP3-18 on neurite growth was rescued by co-expression with Rab1, supporting the conclusion that GTRAP 3-18 acted by inhibiting Rab1 action. Finally, we hypothesized that expression of GTRAP3-18 in the brain shoul be lower at stages of active synaptogenesis compared to early developmental stages. This was the case as expression of GTRAP3-18 declined from E17 to P0 and adult rat brains. Thus, we propose a model where protein trafficking and neuronal differentiation are directly linked by the interaction of Rab1 and its regulator GTRAP3-18.

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GTRAP3-18 slows ER to Golgi transport of VSVG-ts045. (A) A representative example of a single Western blot of VSVG-ts045-YFP and CFP (as a control) or CFP-GTRAP3-18 after synchronization at 40°C overnight, incubation at 32°C for indicated time-points and Endoglycosidase H digest. Lower gel bands represent EndoH sensitive ER-localized fractions and higher bands represent EndoH resistant Golgi-localized fractions. (B) Band intensities of six independent experiments were quantified and the Golgi-accumulation over time was calculated as explained in Materials and methods. Statistical significance was obtained in a paired, two-tailed t-test. Symbols show mean ± S.E.M.
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fig02: GTRAP3-18 slows ER to Golgi transport of VSVG-ts045. (A) A representative example of a single Western blot of VSVG-ts045-YFP and CFP (as a control) or CFP-GTRAP3-18 after synchronization at 40°C overnight, incubation at 32°C for indicated time-points and Endoglycosidase H digest. Lower gel bands represent EndoH sensitive ER-localized fractions and higher bands represent EndoH resistant Golgi-localized fractions. (B) Band intensities of six independent experiments were quantified and the Golgi-accumulation over time was calculated as explained in Materials and methods. Statistical significance was obtained in a paired, two-tailed t-test. Symbols show mean ± S.E.M.

Mentions: Co-expression with GTRAP3-18 reduces the plasma membrane expression of several membrane proteins [19]. To clarify the influence of GTRAP3-18 on trafficking in the early secretory pathway we employed an ER-to-Golgi transport assay. We used the YFP-tagged version of a temperature sensitive mutant of VSVG (VSVG-ts045) as cargo protein. This protein is misfolded at 40°C and retained in the ER. After a temperature shift to 32°C, VSVG-ts045 rapidly folds and enters the secretory pathway. Its presence in the ER or the Golgi can easily be assessed by testing for its sensitivity to EndoH. When the protein reaches the Golgi it becomes resistant to Endo H. We transfected HEK293 cells with a plasmidencoding YFP-tagged VSVG-ts045 and either a plasmid encoding for CFP or a plasmid encoding for CFP-tagged GTRAP3-18. Cells were maintained over night at 40°C to synchronize VSVG-ts045 in the ER. As shown in Fig. 2A, co-expression of CFP-GTRAP3-18 noticeably slowed the transport kinetics. A densitometric quantification of six independent experiments revealed a statistically significant difference between the pooled paired determinants of the control and the GTRAP3-18 group (Fig. 2B, t= 4.057; P< 0.001 in a two-tailed, paired t-test).


GTRAP3-18 serves as a negative regulator of Rab1 in protein transport and neuronal differentiation.

Maier S, Reiterer V, Ruggiero AM, Rothstein JD, Thomas S, Dahm R, Sitte HH, Farhan H - J. Cell. Mol. Med. (2008)

GTRAP3-18 slows ER to Golgi transport of VSVG-ts045. (A) A representative example of a single Western blot of VSVG-ts045-YFP and CFP (as a control) or CFP-GTRAP3-18 after synchronization at 40°C overnight, incubation at 32°C for indicated time-points and Endoglycosidase H digest. Lower gel bands represent EndoH sensitive ER-localized fractions and higher bands represent EndoH resistant Golgi-localized fractions. (B) Band intensities of six independent experiments were quantified and the Golgi-accumulation over time was calculated as explained in Materials and methods. Statistical significance was obtained in a paired, two-tailed t-test. Symbols show mean ± S.E.M.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823040&req=5

fig02: GTRAP3-18 slows ER to Golgi transport of VSVG-ts045. (A) A representative example of a single Western blot of VSVG-ts045-YFP and CFP (as a control) or CFP-GTRAP3-18 after synchronization at 40°C overnight, incubation at 32°C for indicated time-points and Endoglycosidase H digest. Lower gel bands represent EndoH sensitive ER-localized fractions and higher bands represent EndoH resistant Golgi-localized fractions. (B) Band intensities of six independent experiments were quantified and the Golgi-accumulation over time was calculated as explained in Materials and methods. Statistical significance was obtained in a paired, two-tailed t-test. Symbols show mean ± S.E.M.
Mentions: Co-expression with GTRAP3-18 reduces the plasma membrane expression of several membrane proteins [19]. To clarify the influence of GTRAP3-18 on trafficking in the early secretory pathway we employed an ER-to-Golgi transport assay. We used the YFP-tagged version of a temperature sensitive mutant of VSVG (VSVG-ts045) as cargo protein. This protein is misfolded at 40°C and retained in the ER. After a temperature shift to 32°C, VSVG-ts045 rapidly folds and enters the secretory pathway. Its presence in the ER or the Golgi can easily be assessed by testing for its sensitivity to EndoH. When the protein reaches the Golgi it becomes resistant to Endo H. We transfected HEK293 cells with a plasmidencoding YFP-tagged VSVG-ts045 and either a plasmid encoding for CFP or a plasmid encoding for CFP-tagged GTRAP3-18. Cells were maintained over night at 40°C to synchronize VSVG-ts045 in the ER. As shown in Fig. 2A, co-expression of CFP-GTRAP3-18 noticeably slowed the transport kinetics. A densitometric quantification of six independent experiments revealed a statistically significant difference between the pooled paired determinants of the control and the GTRAP3-18 group (Fig. 2B, t= 4.057; P< 0.001 in a two-tailed, paired t-test).

Bottom Line: The effects on the early secretory pathway in HEK293 cells were: reduction of the rate of ER-to-Golgi transport of the vesicular stomatitis virus glycoprotein (VSVG), slowed accumulation of a Golgi marker plasmid in pre-Golgi structures after Brefeldin A treatment and inhibition of cargo concentration of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) into transpor complexes in HEK293 cells, an effect that could be completely reversed in the presence of an excess of Rab1.The inhibitory effect of GTRAP3-18 on neurite growth was rescued by co-expression with Rab1, supporting the conclusion that GTRAP 3-18 acted by inhibiting Rab1 action.Finally, we hypothesized that expression of GTRAP3-18 in the brain shoul be lower at stages of active synaptogenesis compared to early developmental stages.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology, Center for Biomolecular Medicine and Pharmacology, Medical University Vienna, Waehringer Strasse, Vienna, Austria.

ABSTRACT
Glutamate transporter associated protein 3-18 (GTRAP3-18) is an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family interacting with small Rab GTPases, which regulate intracellular trafficking events. Its impact on secretory trafficking has not been investigated. We report here that GTRAP3-18 has an inhibitory effect on Rab1, which is involved in ER-to-Golg trafficking. The effects on the early secretory pathway in HEK293 cells were: reduction of the rate of ER-to-Golgi transport of the vesicular stomatitis virus glycoprotein (VSVG), slowed accumulation of a Golgi marker plasmid in pre-Golgi structures after Brefeldin A treatment and inhibition of cargo concentration of the neuronal glutamate transporter excitatory amino-acid carrier 1 (EAAC1) into transpor complexes in HEK293 cells, an effect that could be completely reversed in the presence of an excess of Rab1. In accordance with the known role of Rab1 in neurite formation, overexpression of GTRAP3-18 significantly inhibited the length of outgrowing neurites in differentiated CAD cells. The inhibitory effect of GTRAP3-18 on neurite growth was rescued by co-expression with Rab1, supporting the conclusion that GTRAP 3-18 acted by inhibiting Rab1 action. Finally, we hypothesized that expression of GTRAP3-18 in the brain shoul be lower at stages of active synaptogenesis compared to early developmental stages. This was the case as expression of GTRAP3-18 declined from E17 to P0 and adult rat brains. Thus, we propose a model where protein trafficking and neuronal differentiation are directly linked by the interaction of Rab1 and its regulator GTRAP3-18.

Show MeSH
Related in: MedlinePlus