Research ArticleTransport of mannose-6-phosphate receptors from the trans-Golgi network to endosomes requires Rab31
Introduction
Intracellular membrane trafficking is essential to maintain both the structural and functional organization of cells. An elaborate system of vesicle–tubular carriers is employed in communication among the compartments of the exocytic and endocytic pathways and the plasma membrane. This membrane transport governs the composition and size of the various cellular compartments and cell surface [1].
Membrane transport pathways are regulated by the concerted control of: 1) the formation and release of carriers, 2) the movement of carriers along elements of the cytoskeleton, and 3) the targeting and fusion of carriers [2], [3]. Rab proteins are key components in these mechanisms of carrier formation, movement and carrier targeting [4]. The family of Rab proteins encompasses more than 70 members; each member regulates a specific vesicle trafficking pathway [50]. For example, Rab1 is involved in transport from the endoplasmic reticulum to the Golgi complex [5], Rab8 in transport from the trans-Golgi network (TGN) to the plasma membrane [6], Rab3 in transport from synaptic vesicles to the plasma membrane [7], Rab5 in transport from the plasma membrane to early endosomes [8], Rab6 in transport from Golgi to endoplasmic reticulum [9], Rab7 in transport from late endosomes to lysosomes [10], and Rab 9 in transport from late endosomes to the TGN [11].
Despite numerous studies, the role of many Rab proteins remains unknown. A novel Rab protein we cloned from a rat oligodendrocyte cDNA library was designated rRab22b, because of its similarity to human Rab22b [12]. Its high degree of homology, 96%, with human Rab22b indicates that rRab22b is the orthologue of human Rab22b. Subsequent publications by others identify Rab22b as Rab31 [13]. For consistency with the literature, we now refer to rRab22b as Rab31.
In a previous study, we found that Rab31 is localized in the TGN and endosomes. Rab31 partially co-localized with both early endosomal markers Rab5 and endocytosed transferrin (after incubation of cells for 5 min with Texas Red transferrin) [12]. The presence of Rab31 in TGN and early endosomes was recently corroborated by two other groups [14], [15]. Indeed, Rab31 co-localized with syntaxin-6, a marker for TGN, and partially co-localized with EEA1, a marker for early endosomes.
We showed by time-lapse video microscopy that Rab31 localized in tubulo vesicular carriers that bud from the TGN, travel along microtubules toward the cell periphery, and fuse with endocytic compartments [12]. These observations suggest the involvement of Rab31 in TGN to endosome transport. However, the nature and biological significance of the Rab31 dependent membrane transport remain unknown.
Two distinctive membrane transport pathways transport newly synthesized proteins from the TGN to endocytic compartments. One pathway depends on both cation-dependent and cation-independent MPRs and transport lysosomal enzymes. Lysosomal hydrolases are transported to endosomes in a complex with MPRs [16]. In endosomes the complex dissociates, the hydrolases are delivered to the lysosomes, and the MPRs are then recycled to the TGN [17]. MPR–hydrolase complexes are transported from the TGN to endosomes in carriers coated with clathrin and the gamma ear containing, ADP ribosylation factor binding proteins (GGAs) [18], [19]. GGAs contribute to the recruitment of the MPR–hydrolase complexes to new carriers through interaction between the GGA1 VHS domain and the DXXLL signal in the cytosolic domain of MPRs [20].
The other pathway does not depend on MPRs and transports newly synthesized lysosome-associated membrane proteins (LAMPs), including LAMP-1, LAMP-2 and LAMP-3 (also known as CD63), [21], [22], [23]. CD63 is transported from TGN to lysosomes through a route involving passage through late endosomes. However, a fraction of newly synthesized CD63 may move from TGN to early endosomes before being targeted to lysosomes, and also via the plasma membrane [24]. Sorting of CD63 into carriers budding from the TGN appears to be mediated by the signal GYEVM that is present in its cytosolic domain.
In this study, we demonstrated the involvement of Rab31 in the transport of MPRs from the TGN to endosomes. Analysis by double fluorescence microscopy of HeLa cells co-expressing Rab31-EYFP and CD-MPR-ECFP show that CD-MPR-ECFP in the TGN is specifically sorted to carriers containing Rab31-EYFP. In contrast, newly synthesized CD63-EYFP and VSVG-EYFP are not sorted to Rab31 carriers. Our results also show that two coat proteins, clathrin light chain b isoform and GGA1, co-localized with Rab31 in the TGN, carriers and peripheral tubulo vesicular structures. Expression of the mutant Rab31(S19N) inhibited the formation in the TGN of carriers containing CD-MPR. In contrast, expression of the mutant Rab31(Q64L) increased slightly the formation of carriers containing CD-MPR. Depletion of endogenous Rab31 causes collapse of the Golgi apparatus. This study indicates that Rab31 participates in both the transport of MPRs from TGN to endosomes and in the Golgi/TGN organization.
Section snippets
Recombinant DNA procedures
The cloning of Rab31 tagged with EYFP or ECFP was described previously [12]. Rab31(S19N) and Rab31 (Q64L)mutants were obtained by site-directed mutagenesis according to the method of Ho et al. [25].
The CD63-ECFP, CD63-EYFP, clathrin-ECFP, GGA1-ECFP and CD-MPR-ECFP expression plasmids were provided by Dr. Juan Bonifacino (Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA). The VSVG-Venus expression
Results
Rab proteins localize to the sites at which they regulate the traffic events. Our previous studies showed that Rab31 is present in the TGN, in peripheral small tubular vesicles and endosomes, suggesting that Rab31 is involved in the traffic of membranes between TGN and endosomes [12]. However, to define its role it is necessary to establish which cargo proteins are transported through the Rab31 pathway.
Indeed, from the TGN, newly synthesized proteins are targeted to endocytic compartments
Discussion
In this paper we show that Rab31 is part of the molecular mechanisms that regulate the transport of MPRs from the TGN to endosomes. Our results suggest that Rab31 participates in the formation of carrier vesicles in the TGN. Our observations also show that Rab31 is also involved in maintaining the structure of the Golgi/TGN.
The MPR transport pathway is used to transport newly synthesized endosomal and lysosomal proteins from the TGN to endosomes. This pathway is also traveled by certain
Acknowledgments
We thank Dr. Maurice Rapport and Dr. Teresa DiLorenzo for editing and commenting on the article. We thank Mr. Michael Cammer, Director of light Microcopy and Image Analysis, for his valuable help in the processing of the cell images. This work was supported by NIH grant RO1 NS 47500-01 to J.N.L. Y. X. and S. Q. were supported by NIH training grant NS 07098.
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