Gene symbols: Rab6a, Rab6b

1. General Function

Rab6 proteins are the mammalian homolog of yeast Ypt6.  There are three forms: 6a, 6a’ (also known as 6c) and 6b (6).  Rab6a and 6a’ are splice variants differing in only 3 amino acids and are expressed in most tissues.  Rab6b is expressed mainly in brain (14).  Rab6 localizes primarily to the Golgi apparatus with increasing amounts in the trans Golgi and trans Golgi network (TPN).  As with other Rab proteins, Rab6 is geranylgeranylated on two cysteine residues at its carboxy terminal and this prenylation is essential for its localization to cellular membranes.  Little is known of GEFs that activate Rab6 although two GAPs with activity towards Rab6 are known (6).

Functionally Rab6 is believed to be involved in a type of retrograde intra-Golgi transport that is independent of the primary form which utilizes COP I (6,17).  Evidence suggests that Rab6a regulates the link between membranes and microtubule-based motor proteins.  A second potential function for Rab6 is the budding off from the TGN of constitutive exocytotic vesicles which associate with kinesin 1 and move to the plasma membrane (9).  Recently Rab 6 and 6a were shown to recruit myosin II to induce fission of vesicles from tubules that bud off the TGN (13)There is also evidence that Rab6a’ is involved in transport from early/recycling endosomes to the TGN (11).  A plethora of potential Rab6 effector proteins have been identified with many being either Golgins or kinesins (1,3,16) most of which bind to Rab6 in the GTP-liganded form.  Both Rab6a and 6a’ have two PKC phosphorylation sites and phosphorylation may affect their membrane association (8).  Rab 6 has been crystallized and its structure solved (2).

2. Rab6 in Pancreas

Rab6 has been reported on pancreatic zymogen granules by Western blotting (10) and mass spectrometry (5,15).  In one of these studies it was reported as Rab6a (14).  When immunohistochemistry was used, however, only a small fraction of isolated ZG were stained and the primary cellular localization was on the Golgi and TGN (5).  As shown in the figure below Rab6 staining is largely overlapping with TGN38 staining, a marker for the TGN.

Rab6 has also been studied in rat AR42J and human SOJ-6 pancreatic cell lines (4).  Rab6 mRNA and protein were identified in both cells and the localization of Rab6 protein was primarily over the Golgi by immunohistochemistry.  In AR42J cells Rab6 partioned between cytosol and microsomal membrane fractions.  However, in SOJ-6 cells it was only present associated with membranes.  The authors showed that different forms of Rab GDI were absent or altered in SOJ-6 cells and suggested that these changes could underly the failure of SOJ-6 cells to secrete Bile Salt Dependent Lipase (BSDL).  In AR42J cells treatment with antisense oligonucleotide specific for Rab6 decreased BSDL secretion after 70 hours indicating that Rab6 is involved in the secretory process in AR42J cells (4).

3. Tools for study of Rab6

a. cDNA

cDNA clones for human Rab6a, 6b, and 6a’ (6c) in pcDNA 3.1 are available from the Missouri S & T cDNA Resource (www.cdna.org/).  Several studies have been published using constitutively active (Rab6 Q72L) or dominant negative (Rab6 T27N) mutant plasmids based on mutating residues known to be important in Ras (7,12). 

b. Rabbit antibodies

Rabbit antibodies raised against the C-terminal of human Rab6 (Ser 184 to Glu203 have been used to identify Rab6 by western blotting and immunohistochemistry in AR42J cells (4) and Rat pancreas (5).  A Ab against this region is sold by Santa Cruz Biotechnology as a IgG (sc-310) and stated to detect all isoforms of Rab6 of mouse, rat, and human origin.  We used this Ab for immunohistochemistry (Fig 1) at a dilution of 1:50 -1:100 (5).  A mouse monoclonal against Rab6a is available from Abcam (ab55660) but has not been checked by us on pancreas.

c. Mouse lines

None.

Figure 1. Confocal immunohistochemistry supplied by S.A. Ernst (University of Michigan) and was carried out on frozen sections of isolated rat acini with rabbit Anti-Rab6 (red) from Santa Cruz (sc-310) and mouse monoclonal Ab against TGN38 (green) from Abcam (ab2809). DAPI (blue) was used to stain nuclei. In the bottom right panel the fluorescence is superimposed on a Nomarski Image.

4. References

1. Barr FA. A novel Rab6-interacting domain defines a family of Golgi-targeted coiled-coil proteins. Curr Biology 9:381-384, 1999. PMID: 10209123
2. Bergbrede T, Pylypenko O, Rak A, Alexandrov K. Structure of the extremely slow GTPase Rab6a in the GTP bound form at 1.8A resolution.  J Struct Biol. 152:235-238, 2005. PMID: 16332443
3. Bergbrede T, Chuky N, Schoebel S, Blankenfeldt W, Geyer M. Fuchs E, Goody RS, Barr F, Alexandrov K. Biophysical Analysis of the interaction of Rab6a GTPase with its effector domains.  J Biol Chem 284: 2628-2635, 2009. PMID: 19019823
4. Caillol N, Pasqualini E, Lloubes R, Lombardo D. Impairment of bile salt-dependent lipase secretion in human pancreatic tumoral SOJ-6 cells.  J Cellular Biochemistry  79:628-647. PMID: 10996854
5. Chen X, Walker AK, Strahler JR, Simon ES, Tomanicek-Volk S, Nelson BB, Hurley MC, Ernst AS, Williams JA, Andrews PC. Organellar Proteomics Analysis of pancreatic zymogen granule membranes. Mol Cell Proteomics. 5:306-312, 2006. PMID: 16278343
6. Draper R.K. Rab6a.  UCSD-Nature Molecule Pages.  Published online 16 May 2007 DOI:10.1038/mp.a001995.01.
7. Echard A, Jollivet F, Martinez O, Lacapere JJ, Rousselet A, Janoueix-Lerosey J, Goud B. Interaction of a Golgi-associated kinesin-    like protein with Rab6.  Science  279:580-585, 1998. PMID: 9438855
8. Fitzgerald ML, Reed GL. Rab6 is phosphorylated in thrombin-activated platelets by a protein kinase C-dependent mechanism: effects on GTP/GDP binding and cellular distribution.  Biochem J. 342:353-360, 1999. PMID: 10455022
9. Grigoriev I, Splinter D, Keijzer N, Wulf PS, Demmers J, Ohtsuka T, Modesti M, Maly IV, Grosveld F, Hoogenraad CC, Akhmanova A. Rab6 regulates transport and targeting of exocytotic carriers.  Dev. Cell.  13:305-314, 2007. PMID: 17681140
10. Iida H, Tanaka S, Shibata Y. Small GTP-binding protein, Rab6, is associated with secretory granules in atrial myocytes.  Am J Physiol 272:C1594-C1601, 1997. PMID: 9176151
11. Mallard F, Tang BL, Galli T, Tenza T, Saint-Pol A, Yue X, Antony C, Hong W, Gould B, Johannes L. Early/recycling endosomes-to-TGN transport involves two SNARE complexes and a Rab6 isoform.  J Cell Biology 156:653-664, 2002. PMID: 11839770
12. Martinez O, Antony C, Pehau-Arnaudet G, Berger EG, Salamero J, Goud B. GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.  Proc Natl Acad Sci USA  94:1828-1833, 1997. PMID: 9050864
13. Miserey-Lenkei S, Chalancon G, Bardin S, Formstecher E, Goud B, Echard A. Rab and actomyosin-dependent fission of transport vesicles at the Golgi complex.  Nature Cell Biology  12:645-654, 2010.  PMID:  20562865
14. Opdam FJ, Echard A, Croes HJ, van den Hurk JA, van de Vorstenbosch RA, Ginsel LA, Goud B, Fransen J. The small GTPase Rab6B, a novel Rab6 subfamily member, is cell-type specifically expressed and localized to the Golgi apparatus.  J Cell Sci  113: 2725-2735, 2000. PMID: 10893188
15. Rindler MJ, Xu C-F, Gumper I, Smith NN, Neubert TA. Proteomic analysis of pancreatic zymogen granules: identification of new granule proteins.  J Proteome Res.  6:2978-2992, 2007. PMID: 17583932
16. Short B, Preisinger C, Schaletzky J, Kopajtich R, Barr FA. The Rab6 GTPase regulates recruitment of the dynactin complex to golgi membranes.  Curr Biology 12:1792-1795, 2002. PMID: 12401177
17. Wanschers B, Van de Vorstenbosch R, Wijers M, Wieringa B, King SM, Fransen J. Rab6 family proteins interact with the dynein light chain protein DYNLRB1.  Cell Motil.  Cytoskeleton. 65:183-196, 2008. PMID: 18044744