Rab13 Traffics on Vesicles Independent of Prenylation

Maria S Ioannou,Martine Girard,Peter S Mcpherson

doi:10.1074/jbc.m116.722298

Maria S Ioannou, Martine Girard + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m116.722298

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Abstract

Rab GTPases are critical regulators of membrane trafficking. The canonical view is that Rabs are soluble in their inactive GDP-bound form, and only upon activation and conversion to their GTP-bound state are they anchored to membranes through membrane insertion of a C-terminal prenyl group. Here we demonstrate that C-terminal prenylation is not required for Rab13 to associate with and traffic on vesicles. Instead, inactive Rab13 appears to associate with vesicles via protein-protein interactions. Only following activation does Rab13 associate with the plasma membrane, presumably with insertion of the C-terminal prenyl group into the membrane.

Highlights

As for all small GTPases, Rabs cycle between an active GTPbound form and an inactive GDP-bound form
Inactive Rab13 Traffics on Vesicles—It is generally assumed that Rabs are soluble in their inactive GDP-bound form and that only upon activation and conversion to the GTP-bound form do they associate with membranes
We show that Rab13 traffics on vesicles derived from both recycling and late endosomal compartments in both its inactive and active form, whereas only active Rab13 is found on the plasma membrane

Summary

Experimental Procedures

Antibodies, DNA Constructs, and shRNA—Mouse monoclonal antibodies used were as follows: Rab and flotillin (BD Biosciences), Rab (Abcam; Cambridge, MA), Naϩ/Kϩ ATPase (Upstate Biotechnology, Lake Placid, NY), and actin (Chemicon). At 18 h post-transfection, cells were lysed in binding buffer (50 mM HEPES (pH 8.0), 300 mM NaCl, 20 mM imidazole, 1% Triton X-100, and protease inhibitors), centrifuged at 200,000 ϫ g for 15 min at 4 °C, and incubated with nickel-nitrilotriacetic acid-agarose beads (Qiagen) with rocking for 2 h at 4 °C. For GDI extraction, HEK-293T cells were treated as described above, and P1 was resuspended in HEPES buffer containing increasing concentrations of purified Hismyc-Rab GDI, incubated for 30 min at 37 °C, and centrifuged at 200,000 ϫ g for 30 min at 4 °C, yielding the S2 and P2 fractions. For co-localization analysis, MCF10A cells were plated on poly-L-lysine-coated coverglass, transfected using JetPrime reagent, and incubated for 14 h. Comparisons of groups were performed using Student’s t test or one-way analysis of variance and Dunnett post-test for multiple comparisons (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001)

Results and Discussion

D MCF10A

Conclusion