A role for Rab30 in retrograde trafficking and maintenance of endosome-TGN organization

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Highlights

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    Rab30 localised primarily to TGN and recycling endosomes.

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    Rab30 plays a major role in the spatial organization of the TGN and recycling endosomes.

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    Deficiency of Rab30 results in a perturbation in endosome-TGN retrograde transport.

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    Interactive partners of Rab30 include Arf1 and Arf4.

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    Findings highlight the interconnected network between the TGN and endosomal system.

Abstract

Rab30 is a poorly characterized small GTPase. Here we show that Rab30 is localised primarily to the TGN and recycling endosomes in a range of cell types, including primary neurons; minor levels of Rab30 were also detected throughout the Golgi stack and early endosomes. Silencing of Rab30 resulted in the dispersal of both early and recycling endosomes and TGN compartments in HeLa cells. By analyzing cargo trafficking in Rab30-silenced and Rab30-overexpressing HeLa cells, we demonstrate that Rab30 plays a role in retrograde trafficking of TGN38 from endosomes to the Golgi, but has no apparent role in the endocytic recycling of the transferrin receptor to the plasma membrane. Five interactive partners with Rab30 were identified by pull-down and MS analysis using GFP-tagged Rab30 mutant, Rab30(Q68L). Two of the interactive partners identified were Arf1 and Arf4, known regulators of endosome to TGN retrograde transport. Knockdown of Arf1 and Arf4 results in GFP-Rab30 decorated tubules arising from the recycling endosomes, suggesting association of Rab30 with tubular carriers. Overall our data demonstrates a role for Rab30 in regulating retrograde transport to the TGN and maintenance of endosomal-TGN organization.

Introduction

Rab30 has the typical characteristics of other Rab proteins, with a size of 23 kDa and the ability to exchange GTP and GDP thereby acquiring two different states of activity [1]. RAB30 was originally discovered from cDNA extracted from human melanocytes and was mapped to chromosome 11 [1,2]. The RAB30 sequence contains the generic isoprenylation sequence (CCAAX) allowing for physical attachment to the membrane bilayer through a geranylgeranyl lipid anchor [1]. Rab30 has been shown to be required for embryo development in Drosophila melanogaster [3], although the function of this small G protein still remains unclear.

Previous studies using immunofluorescence and electron microscopy reported that Rab30 is localised primarily to the Golgi complex in human and Drosophila cells [2,4]. Rab30 was also found to interact in vitro with members of the coiled-coil golgin family from immunoprecipitation and immunoblotting analysis of cytosolic fractions of Drosophila S2 cells [2,4,5]. The golgin interacting partners include, the cis-golgins, GM130 and GMAP210 and the trans-Golgi network (TGN) golgins, golgin-97, golgin-245/p230, GCC88 and GCC185 [4,6]. Further analysis using yeast two-hybrid assays located putative binding sites for Rab30 along the coiled-coil motifs of the golgins [4]. Notably the TGN golgin GCC88 had two binding sites for Rab30. In a separate study, the mRNA binding protein ElF-5A2 was identified by the yeast two-hybrid assay as another putative binding partner for Rab30 [7]. ElF-5A2 is preferentially expressed in the brain and testes and is upregulated in ovarian and colorectal cancer cell lines [8,9]. However, the functional significance of these interactions with Rab30 have not been explored in vivo.

Previous reports have suggested that Rab30 functions predominantly in maintaining the morphology and structure of the Golgi apparatus and no defects in trafficking were reported when Rab30 was inactivated [2]. However, other studies have suggested that Rab30 may have roles aside from those in of the Golgi. Ectopic expression of both human and Drosophila Rab30, which share 63% protein sequence identity [10] in Drosophila S2 cells showed that Rab30 was localised to the trans-Golgi compartments as well as vesicles positive for Rab11 [3]. However, the functional significance of Rab30 on Rab11-positive endosomes was not investigated further. Recently, a novel role for Rab30 was reported in regulating the formation of autophagosomes in epithelial cells infected with Group A Streptococcus (GAS) [11]. Rab30 was shown to be actively recruited to GAS-containing autophagosomes-like vacuoles (GcAVs) in response to Streptococcus infection. Collectively, these findings suggest that Rab30 may have roles in endocytic trafficking, in addition to its role in maintaining the integrity of Golgi morphology [11]. Clearly, a more detailed analysis of the location of Rab30 and its role in trafficking is required to resolve the differences in the literature. Here we show that Rab30 is localised not only to the Golgi but also to recycling endosomes and is involved in maintaining the spatial organization of the TGN-recycling endosome interface and in retrograde transport to the TGN.

Section snippets

Plasmids

pEGFP-Rab30WT/pEGFP-Rab30Q68L/pEGFP-Rab30T71N plasmids were constructed by insertion of Rab30WT/Rab30Q68L/Rab30T71N into pEGFP-C1 vector (Clontech, USA) and were verified by DNA sequencing. pIRES-TGN38WT was constructed by cloning untagged TGN38WT into pIRES previously [12]. Ds-Red2-ER construct was purchased from Clontech (USA).

siRNAs

siRNA target sequences were purchased from Sigma-Aldrich (Australia) and are described below.

RNAiSequence (5’ – 3′)Source/Reference
Rab30-siRNA-2CUGUGUUAGUGGGCAACAA

GFP-Rab30Q68L localises to the Golgi complex, early endosomes and recycling endosomes

To characterise Rab30, HeLa cells stably expressing GFP-Rab30Q68L were generated as the available commercial antibody to Rab30 has a significantly low signal to noise ratio and the Rab30 protein was only able to be detected by the commercial antibody when overexpressed (data not shown). The Q68L mutant of Rab30 represents the constitutively active form of Rab proteins (locked in the GTP form) [24]. A polyclonal GFP-Rab30Q68L cell line was established with >80% of cells GFP-positive and then

Discussion

In this paper we have examined the intracellular localisation of Rab30 and the roles of Rab30 at the interface between the recycling endosomes and the TGN. Our findings show that Rab30 is localised predominantly to the TGN and recycling endosomes in a variety of different cell types. Furthermore, silencing of Rab30 in HeLa cells resulted in alterations in the morphology of the TGN as well the intracellular distribution of the early and recycling endosomes. Modification to the levels of cellular

Credit author statement

Khalisah L. Zulkefli: Conceptualization; Formal analysis; Investigation; Methodology; Writing - review & editing. Ismail Mahmoud: Performed proteomics and analysis. review & editing manuscript. Nicholas Williamson: Supervision of proteomics and mass spectrometry analysis, review & editing manuscript. Prajakta Kulkarni Gosavi: Performed and analysed immunogold EM, review & editing manuscript. Fiona J. Houghton: generated and analysed stable cell clones, methodology, performed analyses for Fig.

Acknowledgements

Confocal Microscopy was performed at the Biological Optical Microscopy Platform (BOMP), University of Melbourne, electron microscopy at the Advanced Microscopy Facility, Bio21 Institute, University of Melbourne and cell sorting at the Murdoch Children's Research Institute Flow Cytometry and Imaging Facility. We thank Bruno Goud (Institut Curie, Paris) for Rab30 constructs and Isabelle Gasnereau (Gleeson laboratory) for cloning. This work was supported by funding from the Australian Research

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    • 1

    Present address: Department of Biosciences, University of Oslo, Blindernveien 31, 0371 Oslo, Norway.

    2

    Present address: Department of Medical Laboratory Sciences, The Hashemite University, Zarqa, Jordan.

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