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Arfaptin-1 negatively regulates Arl1-mediated retrograde transport.

Huang LH, Lee WC, You ST, Cheng CC, Yu CJ - PLoS ONE (2015)

Bottom Line: Exogenous arfaptin-1 expression did not interfere with the localization of the Arl1-interacting proteins golgin-97 and golgin-245 to the TGN and vice versa.Moreover, we found that the N-terminal region of arfaptin-1 was involved in the regulation of retrograde transport.Our results show that arfaptin-1 acts as a negative regulator in Arl1-mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.

ABSTRACT
The small GTPase Arf-like protein 1 (Arl1) is well known for its role in intracellular vesicular transport at the trans-Golgi network (TGN). In this study, we used differential affinity chromatography combined with mass spectrometry to identify Arf-interacting protein 1b (arfaptin-1b) as an Arl1-interacting protein and characterized a novel function for arfaptin-1 (including the arfaptin-1a and 1b isoforms) in Arl1-mediated retrograde transport. Using a Shiga-toxin subunit B (STxB) transportation assay, we demonstrated that knockdown of arfaptin-1 accelerated the retrograde transport of STxB from the endosome to the Golgi apparatus, whereas Arl1 knockdown inhibited STxB transport compared with control cells. Arfaptin-1 overexpression, but not an Arl1 binding-defective mutant (arfaptin-1b-F317A), consistently inhibited STxB transport. Exogenous arfaptin-1 expression did not interfere with the localization of the Arl1-interacting proteins golgin-97 and golgin-245 to the TGN and vice versa. Moreover, we found that the N-terminal region of arfaptin-1 was involved in the regulation of retrograde transport. Our results show that arfaptin-1 acts as a negative regulator in Arl1-mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.

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The effect of arfaptin-1b serine 132 phosphorylation on STxB endocytic transport.HeLa cells were transfected with arfaptin-1a-S100A, arfaptin-1a-S100D, arfaptin-1b-S132A, or arfaptin-1b-S132D for 48 h. A STxB transport assay was conducted with incubation at 37°C for 30 min, followed by staining with anti-myc and anti-GM130 antibodies. Scale bars, 10 μm. Asterisks indicate the exogenous expression of myc-tagged arfaptin-1 constructs. The percentage of cells exhibiting inhibited STxB transport was quantified (n>50 for each experiment), and the data are presented as the means±SDs; p<0.05 indicates significance, as determined by one-way ANOVA.
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pone.0118743.g006: The effect of arfaptin-1b serine 132 phosphorylation on STxB endocytic transport.HeLa cells were transfected with arfaptin-1a-S100A, arfaptin-1a-S100D, arfaptin-1b-S132A, or arfaptin-1b-S132D for 48 h. A STxB transport assay was conducted with incubation at 37°C for 30 min, followed by staining with anti-myc and anti-GM130 antibodies. Scale bars, 10 μm. Asterisks indicate the exogenous expression of myc-tagged arfaptin-1 constructs. The percentage of cells exhibiting inhibited STxB transport was quantified (n>50 for each experiment), and the data are presented as the means±SDs; p<0.05 indicates significance, as determined by one-way ANOVA.

Mentions: Arfaptin-1b acts as a negative regulator of Arf1-mediated vesicle budding [21]. In this study, we identified a potential function for arfaptin-1 in the endocytic pathway and confirmed the role of serine 132 in localization to the Golgi. We next questioned whether the arfaptin-1 phosphorylation status could also regulate retrograde transport of STxB. As shown in Fig. 6, similar to wild-type arfaptin-1b, arfaptin-1b-S132A inhibited STxB transport after 30 min at 37°C compared with control cells. However, STxB transport in arfaptin-1b-S132D-expressing cells was similar to that in control cells after 30 min at 37°C. Similar results were observed in cells expressing arfaptin-1a-S100A and arfaptin-1a-S100D (Fig. 6). These results suggest that PKD—mediated phosphorylation of arfaptin-1 is involved in the regulation of retrograde transport.


Arfaptin-1 negatively regulates Arl1-mediated retrograde transport.

Huang LH, Lee WC, You ST, Cheng CC, Yu CJ - PLoS ONE (2015)

The effect of arfaptin-1b serine 132 phosphorylation on STxB endocytic transport.HeLa cells were transfected with arfaptin-1a-S100A, arfaptin-1a-S100D, arfaptin-1b-S132A, or arfaptin-1b-S132D for 48 h. A STxB transport assay was conducted with incubation at 37°C for 30 min, followed by staining with anti-myc and anti-GM130 antibodies. Scale bars, 10 μm. Asterisks indicate the exogenous expression of myc-tagged arfaptin-1 constructs. The percentage of cells exhibiting inhibited STxB transport was quantified (n>50 for each experiment), and the data are presented as the means±SDs; p<0.05 indicates significance, as determined by one-way ANOVA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4366199&req=5

pone.0118743.g006: The effect of arfaptin-1b serine 132 phosphorylation on STxB endocytic transport.HeLa cells were transfected with arfaptin-1a-S100A, arfaptin-1a-S100D, arfaptin-1b-S132A, or arfaptin-1b-S132D for 48 h. A STxB transport assay was conducted with incubation at 37°C for 30 min, followed by staining with anti-myc and anti-GM130 antibodies. Scale bars, 10 μm. Asterisks indicate the exogenous expression of myc-tagged arfaptin-1 constructs. The percentage of cells exhibiting inhibited STxB transport was quantified (n>50 for each experiment), and the data are presented as the means±SDs; p<0.05 indicates significance, as determined by one-way ANOVA.
Mentions: Arfaptin-1b acts as a negative regulator of Arf1-mediated vesicle budding [21]. In this study, we identified a potential function for arfaptin-1 in the endocytic pathway and confirmed the role of serine 132 in localization to the Golgi. We next questioned whether the arfaptin-1 phosphorylation status could also regulate retrograde transport of STxB. As shown in Fig. 6, similar to wild-type arfaptin-1b, arfaptin-1b-S132A inhibited STxB transport after 30 min at 37°C compared with control cells. However, STxB transport in arfaptin-1b-S132D-expressing cells was similar to that in control cells after 30 min at 37°C. Similar results were observed in cells expressing arfaptin-1a-S100A and arfaptin-1a-S100D (Fig. 6). These results suggest that PKD—mediated phosphorylation of arfaptin-1 is involved in the regulation of retrograde transport.

Bottom Line: Exogenous arfaptin-1 expression did not interfere with the localization of the Arl1-interacting proteins golgin-97 and golgin-245 to the TGN and vice versa.Moreover, we found that the N-terminal region of arfaptin-1 was involved in the regulation of retrograde transport.Our results show that arfaptin-1 acts as a negative regulator in Arl1-mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.

ABSTRACT
The small GTPase Arf-like protein 1 (Arl1) is well known for its role in intracellular vesicular transport at the trans-Golgi network (TGN). In this study, we used differential affinity chromatography combined with mass spectrometry to identify Arf-interacting protein 1b (arfaptin-1b) as an Arl1-interacting protein and characterized a novel function for arfaptin-1 (including the arfaptin-1a and 1b isoforms) in Arl1-mediated retrograde transport. Using a Shiga-toxin subunit B (STxB) transportation assay, we demonstrated that knockdown of arfaptin-1 accelerated the retrograde transport of STxB from the endosome to the Golgi apparatus, whereas Arl1 knockdown inhibited STxB transport compared with control cells. Arfaptin-1 overexpression, but not an Arl1 binding-defective mutant (arfaptin-1b-F317A), consistently inhibited STxB transport. Exogenous arfaptin-1 expression did not interfere with the localization of the Arl1-interacting proteins golgin-97 and golgin-245 to the TGN and vice versa. Moreover, we found that the N-terminal region of arfaptin-1 was involved in the regulation of retrograde transport. Our results show that arfaptin-1 acts as a negative regulator in Arl1-mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.

Show MeSH