1. General Function
Rab8 (Ras-related proteins in brain 8) is a 24 kDa protein that belongs to the Rab GTPase family (7). Rab proteins are known for their participation in and regulation of intracellular membrane trafficking pathways (17, 45, 47). In their active GTP-bound state different Rab proteins bind to different membrane compartments and recruit specific effector proteins, which are not only involved in docking and fusion with the target membrane but also in the formation of transport vesicles and in binding motor proteins for vesicle transport (17, 18, 27, 44, 47).
Rab8 displays high homology with the yeast S. cerevesiae protein SEC4 involved in post-Golgi traffic (16, 24) and with the S. pombe protein Ypt2p that acts as well in the last stage of the secretory pathway (4). In humans there are two isoforms with 80% homology, Rab8a and Rab8b, which have a differential expression pattern (Figure 1). Rab8b is expressed in spleen, testis and brain (2, 8) while Rab8a is more ubiquitous expressed but shows low expression levels in the three organs (8). Nevertheless, Rab8a and Rab8b have been shown to play a similar role in vesicular traffic from the Golgi complex to the plasma membrane in regulated secretion in ATt20 cells (8). The Rab8 lipid motif differs from other Rabs resulting in only one geranylgeranylation (3, 26). A preliminary X-ray crystallographic analysis of mammalian Rab8 in complex with the nucleotide exchange factor MSS4 has been published (25).
Functionally, Rab8 has been proposed to act as a regulator of post-Golgi membrane traffic from the TGN to the plasma membrane. Rab8 has been localized to the Golgi region, to TGN-derived transport vesicles en route to the plasma membrane (22-24), and has been demonstrated to regulate biosynthetic trafficking pathways from the TGN to the cell surface (1, 24, 34). In C. elegans, depletion of ce-rab-8 inhibited the secretion of the growth factor EGL-17 in vivo (28). Rab8 depletion in developing neurons by antisense oligonucleotides inhibited the formation of exocytic vesicles at the Golgi apparatus and thus, neuronal process outgrowth (23). In addition, Rab8 is involved in the targeting of vesicles to the cilium, promoting the extension of the ciliary membrane (34). Rab8 can also modulate the transport of the adrenergic receptors α2B- and β2- by direct interaction (14).
Recently, it has been demonstrated that Rab8a regulates apical protein localization in intestinal epithelial cells by generating conditional Rab8 knockout mice (42). The mis-localization and degradation of apical peptidases and transporters in lysosomes was observed, thus leading to a marked reduction in the absorption rate of nutrients in the small intestine, and to death. In addition, we have reported on a role of Rab8 in the formation of zymogen granules in pancreatic acinar AR42J cells (15). These data provide evidence for a role of Rab8 in apical trafficking of digestive enzymes in acinar cells of the pancreas.
It has been shown that Rab8 promotes polarized membrane transport through reorganization of actin and microtubules. Rab8 is supposed to modify cellular shape by changing the organization of the plasma membrane and the underlying cytoskeleton (2, 36). Rab8 depletion promotes the formation of actin stress fibers, whereas activation of Rab8 has the opposite effect and causes the formation of cell protrusions (20, 36). Furthermore, Rab8 is supposed to interact via optineurin with myosin VI (41), and was reported to partially associate with mature melanosomes and to regulate actin-based movement of melanosomes (5, 6). Through its interaction with optineurin Rab8 is able to modify epithelial cell shape (21), and in response to apoptotic stimuli Rab8 is involved in the translocation of optineurin to the nucleus (11). It has also been described that the interaction of optineurin with Huntingtin links Huntingtin to Rab8 and promotes the relocalization of Huntingtin to vesicles (12, 48). Already in 1995, it was described that Rab8 may regulate some of the final steps in the post-Golgi transport of rhodopsin in retinal photoreceptor cells (13). Later it was observed that the expression of a Rab8 inactive mutant causes retinal degeneration in Xenopus laevis (32), and more recently that the disruption of the interaction between optineurin and Rab8 leads to degeneration of the retina (10). Another Rab8 interacting protein is Mss4 that assists in nucleotide release (25).
Rab8 has also been linked to the organization of the endocytic compartment, (20). It has been localized to the endosomal recycling compartment (1, 46), where it colocalizes with EHD1 (40) and partially with Rab11 (20). Cells lacking Rab8 are defective in trafficking of the transferrin receptor (35) and display an inhibition of the retrograde transport of cholera toxin B to the Golgi compartment (20). Rab8 plays as well a role in the degradation of cholesterol (29, 30).
2. Specific function in the pancreas
Rab8a has been localized to zymogen granules in acinar cells of the exocrine rat pancreas and in pancreatic AR42J cells using immunocytochemistry (15) (Figure 2). It is present on isolated zymogen granules from rat pancreas and in the granule membrane fraction obtained after granule subfractionation (15). Furthermore, the presence of Rab8a and Rab8b in zymogen granule fractions was reported in recent proteomics studies (9, 38). Silencing of Rab8 (but not of Rab3) by siRNA inhibited granule formation and thus secretion of zymogens in AR42J cells, and resulted in an accumulation of granule marker proteins within the Golgi complex (15). By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide evidence for a role of Rab8 early on in zymogen granule formation at the Golgi complex and thus, apical trafficking of digestive enzymes.
Further evidence for a role of Rab8a in the regulation of apical protein localization has been obtained by the generation of conditional Rab8 knockout mice (42). These mice died 3–4 weeks after birth. In the intestinal epithelial cells of the wild type mice Rab8 localized to the Golgi complex and early endosomes. Interestingly, in the Rab8 knockout mice these cells displayed normal basolateral protein transport, but apical marker proteins accumulated intracellularly in late endosomes/lysosomes and were diminished in the apical membrane (42). The mislocalization and degradation of apical peptidases and transporters in lysosomes is supposed to lead to a marked reduction in the absorption rate of nutrients in the small intestine, and to death.
The small intestines of the knockouts were reported to be swollen and to contain undigested milk (42). Ultrastructural studies did, however, not reveal alterations in the number of zymogen granules at postnatal week 2 when controls and knockout mice were compared. Interestingly, a great reduction in the number of zymogen granules was observed at postnatal week 3, which might be explained by autophagy due to starvation conditions (31) (A. Harada, personal communication). Furthermore, the knockout mice still retain a functional Rab8b isoform, which could compensate for the absence of Rab8a in the pancreas. Rab8 knockout mice show a similar phenotype than patients with human microvillus inclusion disease but only one of the patients studied so far showed a decrease in Rab8 in the small intestine (42).
3. Tools to study Rab8
a. cDNA clones
Different plasmids codifying for the Rab8 wild type and constitutively active and inactive forms have been generated by several laboratories. In 1993, Rab8 was cloned by Huber and coworkers by inserting full-length cDNA into pGEM1 (24). Rab8-pEGFP, pEGFP-Rab8-T22N, pEGFP-Rab8-Q67L, pEGFP-Rab8bwt, pEGFP-Rab8b-T22N, Rab8b-Q67L have been described (19, 21).
pBD-, pmCerulean, pmVenus, and pmCherry versions of wild-type and mutant Rab8a are as well available (40).
The open reading frames of Rab8-T22N and Rab8-Q67L were also cloned into pcDNA4/TO (Invitrogen) and pIRES (Clontech). GST-tagged fusions of Rab8, Rab8-T22N and Rab8-Q67L inserted into pEBG-SrfI have been generated (20). Rab8 and HA-Rab8 in pcDNA3.1 are available at www.cdna.org
For yeast two hybrid experiments Rab8 and its mutant forms (Rab8b-Q67L, Rab8b-T22N, Rab8b-C204S, and Rab8b-N121I) were cloned in the vector pAS2-1 (8). Hattula and coworkers also cloned the Rab8 lacking its carboxy-terminal codons CVLL (Rab8?wt), Rab8, Rab8-67L (activated), Rab8-22N (dominant negative), Rab8?-67L, Rab8?-22N Rab8b, Rab8b-67L, Rab8b-22N into a pGilda vector (21).
For transfection in X. laevis, canine rab8 mutant and wild-type cDNAs (36) were cloned into the vector XOPeGFP-C1 (32, 33).
Purified mouse anti-Rab8 (BD biosciences, 610844) was used successfully for immunoblotting (15, 20, 30, 32, 37), immunohistochemistry of rat pancreas, immunocytochemistry of AR42J cells and immunofluorescence on isolated zymogen granules (15) and other cell types (30, 39).
Recombinant Semliki Forest (SFV) and Adeno (AdV) Viruses have been described (pRab8-SFV, (30), pAdEasy-1, (29)).
d. KO mice
A small-intestine-specific knockout mouse (Rab8geo/geo) and a nullizygous mouse (Rab8-/-) have been described (42).
e. Rab8 silencing
Rab8a expression has been silenced by small interfering RNA (siRNA) (15, 20, 29, 37, 43) or by using short hairpin RNA (shRNA) (14, 30). Rab8b-specific siRNA is also available (30, 43)
Supported by the German Research Foundation (DFG, SCHR 518/5-1, 2), the J. Manchot foundation (Düsseldorf, Germany), the Portuguese Foundation for Science and Technology (FCT) [SFRH/BPD/37725/2007 (to M. G. L), SFRH/BD/48722/2008 (to M. A.)], and the University of Aveiro.
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