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

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.

1

GTRAP3-18 is a resident endoplasmic reticulum (ER) protein and does not interact with Sec24D. (A, B) HEK293 were transfected with plasmids encoding either YFP-tagged wild-type GTRAP3-18 or a mutant with a deletion of the last four amino acids (GTRAPd4) together with a plasmid encoding CFP-ER. Images were acquired 24 hrs after transfection and co-localized using the Zeiss LSM Image Browser software. (C) HEK293 cells expressing YFP-GTRAP3-18 were kept at 15°C for 2 hrs prior to image acquisition. (D) Cytosol (100 μg) prepared from cells overexpressing YFP-Sec24D was co-incubated either with GST, GST-tagged GTRAP3-18 C-terminus or GST-tagged GAT1 C-terminus. GST-pulldown was performed as indicated in Materials and methods. The top panel represents the Ponceau-stained membrane to visualize the loading of the various GST-constructs.

2

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.

3

GTRAP3-18 binds Rab1a in vivo and thus significantly reduces cargo concentration of the EAAC1 glutamate transporter at transport complexes, an effect that can be completely overcome by an excess of Rab1a. (A) Equal amounts of lysates of untransfected or HA-GTRAP3-18 trans-fected HEK293 cells were immunoprecipitated using HA-agarose. Immune complexes were subjected to SDS-PAGE and co-immunoprecipitated, endogenous Rab1a was visualized with an anti-Rab1 antibody. (C) HEK293 cells on coverslips were transfected with CFP-EAAC1 as cargo, Sar1-T39N to synchronize the cargo in the ER and GTRAP3-18 if indicated. Cells were permeabilized after 24 hrs and a concentration assay was performed introducing fresh cytosol (either untransfected or Rab1a enriched cytosol as indicated) and therefore also wild type Sar1 to start ER export. Directly after adding of the ER budding reaction (0 min.; see a in B displaying diffuse staining of EAAC1) as well as after incubation at 37° for 15 min. (see b in B displaying EAAC1 in concentrated complexes) pictures were taken and cells with diffuse staining and with formed transport complexes (TC) were counted. P-values were obtained with an anova followed by Scheffe post-hoc comparisons test). Bars show mean ± S.E.M.

4

GTRAP3-18 slows recovery of YFP-Golgi into pre-Golgi structures after treatment with Brefeldin A. HEK293 cells were transfected with YFP-Golgi alone or together with CFP-tagged GTRAP3-18. After Brefeldin A washout, cells were kept at 37°C and images were acquired at indicated time-points. Cells with diffuse distribution of the marker plasmid (A, a) and with the marker in pre-Golgi intermediates (A, b) were scored. The graph in (B) represents the percentage of marker in pre-Golgi intermediates at 0 min. (a), 15 min. (b) and 45 min. (c) after Brefeldin A washout. P-values were calculated in a paired t-test. Bars show mean ± S.E.M.

5

GTRAP3-18 hinders and Rab1 facilitates neurite outgrowth in differentiated CAD cells. CAD cells were transfected with pEYFP-C1 alone (control) or together with indicated plasmids in a 1: 1 ratio. Cells were then either kept in complete medium or in starving medium lacking FBS to induce differentiation for 20 hrs. Images were acquired and neurite length was measured. A Shows a scatter plot of all measured values per sample (red line represents the mean of all values). The black line represents a cut-off value of 42 μm that was chosen because it accounts for 95% of values in the undifferentiated control groups of the same experiment (YFP alone as control and YFP with CFP-GAT1d37). The box plot in (B) visualizes the data from (A) after log-transformation as median with 25% and 75% percentile as box and minimum and maximum as error bars. P-values were produced with an anova followed by Bonferroni's multiple comparison test. Ns indicates not significant. P-values in black show statistical significance between control and differentiated conditions within the same group, P-values in blue show statistical significance between the differentiated control sample and differentiated samples of all other groups. The percentage of cells with neurites over 42 μm length is shown in (C). Data for the box plot in (D) is obtained as in (B) and includes a subset that is triple-transfected with pEYFP-C1: CFP-Rab1-wt: HA-GTRAP in a 1: 3: 2 ratio. Only cells displaying a CFP- and YFP-signal were included.

6

RNAi-mediated knockdown of GTRAP3-18 has no effect on neurite outgrowth of CAD cells. (A) Knockdown efficiency of two siRNAs against GTRAP3-18 after 48 hrs as determined by semi-quantitative RT-PCR (Inset: Representative agarose gel detail). GTRAP expression levels were corrected for GAPDH levels of the same PCR and the value of the control PCR was set to 100% of expression. A shows a representative level of knockdown for conditions used in all RNAi experiments. B measured neurite length in CAD cells after siRNA-mediated knockdown of GTRAP3-18 and differentiation. For detailed explanation of (B) see corresponding graph in Fig. 5.

7

Expression pattern of GTRAP3-18 during developmental stages that are relevant for neurite outgrowth and synaptogenesis. Total brain RNA of Sprague-Dawley rats of different developmental stages was isolated and subjected to GTRAP and GAPDH double semi-quantitative RT-PCR. A scatter plot shows all measured values for GTRAP3-18 expression after correction for GAPDH levels as arbitrary units (A.U.). The black line represents the mean per sample. P-values were calculated with an anova followed by Bonferroni's multiple comparison test.