Jennifer Navarre

Identification and Characterization of a Family of Rab11-interacting Proteins

Rab11a is a small GTP-binding protein enriched in the pericentriolar plasma membrane recycling systems. We hypothesized that Rab11a-binding proteins exist as downstream effectors of its action. Here we define a family of four Rab11-interacting proteins: Rab11-Family Interacting Protein 1 (Rab11-FIP1), Rab11-Family InteractingProtein 2 (Rab11-FIP2), Rab11-FamilyInteracting Protein 3 (Rab11-FIP3), and pp75/Rip11. All four interacting proteins associated with wild type Rab11a and dominant active Rab11a (Rab11aS20V) as well as Rab11b and Rab25. Rab11-FIP2 also interacted with dominant negative Rab11a (Rab11aS25N) and the tail of myosin Vb. The binding of Rab11-FIP1, Rab11-FIP2, and Rab11-FIP3 to Rab11a was dependent upon a conserved carboxyl-terminal amphipathic α-helix. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 colocalized with Rab11a in plasma membrane recycling systems in both non-polarized HeLa cells and polarized Madin-Darby canine kidney cells. GFP-Rab11-FIP3 also colocalized with Rab11a in HeLa cells. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 also coenriched with Rab11a and H+K+-ATPase on parietal cell tubulovesicles, and Rab11-FIP1 and Rab11-FIP2 translocated with Rab11a and the H+K+-ATPase upon stimulating parietal cells with histamine. The results suggest that the function of Rab11a in plasma membrane recycling systems is dependent upon a compendium of protein effectors.

Rab11b resides in a vesicular compartment distinct from Rab11a in parietal cells and other epithelial cells

The Rab11 family of small GTPases is composed of three members, Rab11a, Rab11b, and Rab25. While recent work on Rab11a and Rab25 has yielded some insights into their function, Rab11b has received little attention. Therefore, we sought to examine the distribution of endogenous Rab11b in epithelial cells. In rabbit gastric parietal cells, unlike Rab11a, Rab11b did not colocalize or coisolate with H(+)/K(+)-ATPase. In MDCK cells, endogenous Rab11b localized to an apical pericentrisomal region distinct from Rab11a. The microtubule agents nocodazole and taxol dramatically alter Rab11as localization in the cell, while effects on Rab11bs distribution were less apparent. These results indicate that in contrast to Rab11a, the Rab11b compartment in the apical region is not as dependent upon microtubules. While Rab11a is known to regulate transferrin trafficking in nonpolarized cells and IgA trafficking in polarized cells, Rab11b exhibited little colocalization with either of these cargoes. Thus, while Rab11a and Rab11b share high sequence homology, they appear to reside within distinct vesicle compartments.

AKAP350 at the Golgi apparatus. II. Association of AKAP350 with a novel chloride intracellular channel (CLIC) family member.

AKAP350 can scaffold a number of protein kinases and phosphatases at the centrosome and the Golgi apparatus. We performed a yeast two-hybrid screen of a rabbit parietal cell library with a 3.2-kb segment of AKAP350 (nucleotides 3611–6813). This screen yielded a full-length clone of rabbit chloride intracellular channel 1 (CLIC1). CLIC1 belongs to a family of proteins, all of which contain a high degree of homology in their carboxyl termini. All CLIC family members were able to bind a 133-amino acid domain within AKAP350 through the last 120 amino acids in the conserved CLIC carboxyl termini. Antibodies developed against a bovine CLIC, p64, immunoprecipitated AKAP350 from HCA-7 colonic adenocarcinoma cell extracts. Antibodies against CLIC proteins recognized at least five CLIC species including a novel 46-kDa CLIC protein. We isolated the human homologue of bovine p64, CLIC5B, from HCA-7 cell cDNA. A splice variant of CLIC5, the predicted molecular mass of CLIC5B corresponds to the molecular mass of the 46-kDa CLIC immunoreactive protein in HCA-7 cells. Antibodies against CLIC5B colocalized with AKAP350 at the Golgi apparatus with minor staining of the centrosomes. AKAP350 and CLIC5B association with Golgi elements was lost following brefeldin A treatment. Furthermore, GFP-CLIC5B-(178–410) targeted to the Golgi apparatus in HCA-7 cells. The results suggest that AKAP350 associates with CLIC proteins and specifically that CLIC5B interacts with AKAP350 at the Golgi apparatus in HCA-7 cells.

23 - Expression and Properties of Rab25 in Polarized Madin-Darby Canine Kidney Cells

This chapter discusses the expression and properties of Rab25 in polarized Madin-Darby canine kidney cells. In Madin-Darby canine kidney (MDCK) cells, Rab25 levels are very low, whereas endogenous Rablla levels are relatively high. Therefore, to study Rab25 in MDCK cells, cell lines are isolated stably transfected with the wild-type sequence of rabbit Rab25. To detect Rab25, monoclonal antibodies are developed specific for Rab25 and nonreactive against Rab11a. A double-screening enzyme-linked immunosorbent assay (ELISA) protocolis developed to isolate monoclonal antibodies against Rab25 that do not react with Rablla. To visualize the three-dimensional distribution of Rab proteins in polarized cells, the stably transfected Rab25- expressing 2A3 cell line is grown to confluence on Transwell clear filters and maintained for three days following confluence. Out of 20 lines screened, six lines were found to contain a transfected Rab25 message. The 2A3 line utilized for characterization of the association of Rablla and Rab259 was one of the two lowest expressing cell lines. To compare the effects of stable overexpression of Rab25 on endogenous message levels, Rab25 and Rablla mRNA levels were compared between mock pCB6-transfected cells and the Rab25-transfected 2A3 line.