Expression and localization of a novel Rab small G protein (Rab38) in the rat lung

Abstract

The Rab small G protein family participates in intracellular vesicle transport, including exocytosis and endocytosis. The cDNA encoding a novel Rab-related small G protein (Rab38) has been cloned from rat lung cDNA library and recorded in GenBank (accession no. M94043). However, the expression and localization of the protein in the lung remains primarily unknown. We produced polyhistidine-tagged recombinant Rab38 and a polyclonal antibody with a synthetic peptide. Immunohistochemistry demonstrated that the protein is specifically localized in alveolar type II cells and in bronchial epithelial cells. In situ hybridization using a digoxygenin-labeled RNA riboprobe clearly showed that the mRNA of the protein is localized in alveolar type II cells and bronchial epithelial cells, especially terminal airway epithelial cells. Western blot and reverse transcriptase-polymerase chain reaction showed distinct expression of the protein and mRNA in isolated alveolar type II cells, but not in alveolar macrophages. The native protein was predominantly hydrophobic and was enriched in a high-density vesicle fraction but was barely detectable in nuclear and lamellar body fractions in alveolar type II cells. Immunofluorescence cytochemistry performed on cultured alveolar type II cells showed that Rab38 distributed extensively in the cytoplasm with a distribution pattern similar to endoplasmic reticulum rather than other subcellular organelles. These results suggest that this novel rab small G protein (Rab38) mediates vesicular transport in terminal airway epithelium.

Figure 1.

Nucleic acid sequence of cDNA and predicted amino acid sequence of rab38. The nucleic acid sequence was downloaded from GenBank (accession no. M94043). The cDNA consists of 633 bp, and the predicted molecular weight of the translated protein is 24 kd. Four consensus amino acid sequences of a small G protein superfamily are boxed. GTPase activity: GDLGVGK and DIAG. GTP/GDP-binding activity: NKCD and ETSAK. Nucleic acid sequences of primers, which were used for PCR after reverse transcription of total extracted RNA leading to the subsequent synthesis of the cRNA probe for in situ hybridization, are underlined.

Figure 2.

Expression of native Rab38 protein in total lung and freshly isolated alveolar type II cells and of the recombinant protein in transfected baculovirus-infected Sf9 cells analyzed by Western blot. Total proteins were extracted with 1% Triton X-114 directly from isolated cells or homogenized rat lungs. Triton X-114 phase separation was performed for an aliquot of alveolar type II cell lysate. Equal amounts of 50 μg protein, except for lane 6 (5 μg protein), were loaded on SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. The membrane was immunoblotted with a rabbit anti-rat Rab38 polyclonal antibody and a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin antibody. Color development was performed in the presence of diaminobenzidine and H₂O₂ for less than 3 minutes. Native Rab38 appeared as a 26-kd band (*) and recombinant Rab38 as a 27-kd band (†). Lane 1: Prestained protein marker. Lane 2: Total lung homogenate. Lane 3: Total type II cell lysate. Lane 4: Water-soluble protein in Triton X-114-partitioning of type II cell lysate. Lane 5: Detergent-extracted protein in Triton X-114-partitioning of type II cell lysate. Lane 6: Recombinant baculovirus-infected Sf 9 cell lysate.

Figure 3.

Expression of rab38 in alveolar type II cells and alveolar macrophages. A: Western blot of cell lysates. Cell lysis and Western blot were performed as described in Figure 2 ▶ . Lane 1: Prestained molecular marker. Lane 2: Alveolar type II cell lysate (35 μg protein). Lane 3: Alveolar macrophage lysate (35 μg protein). B: RT-PCR of rab38 for total RNA extracted from whole lung (10 μg RNA) and the two cell preparations (1 μg RNA). Reverse transcription of first strand DNA was performed with random hexamer primers. Then specific primers for a partial length of rab38 cDNA (top) and for GAPDH (bottom) as a control were used for PCR. The PCR products were electrophoresed in 2% agarose and visualized with ethidium bromide stain. Lane 1: Molecular marker (only for top panel). Lane 2: Whole lung. Lane 3: Alveolar macrophages. Lane 4: Alveolar type II cells.

Figure 4.

a: Immunohistochemistry of native Rab38 protein for rat alveolar tissue. Perfused and excised rat lungs were rapidly frozen in an OCT compound and cut into 5-μm-thick sections. The sections were fixed with 4% paraformaldehyde for 20 minutes and then with acetone for 30 seconds. A polyclonal anti-rat Rab38 antibody (8 μg/ml) was added as the first antibody. For controls, the same concentration of the antibody supplemented with the synthesized polypeptide in 100-fold molar excess was used. Biotin-labeled goat anti-rabbit IgG antibody was added, followed by horseradish peroxidase-conjugated streptavidin. Color development was performed for ∼3 minutes in the presence of diaminobenzidine and H₂O₂. The slides were counterstained with hematoxylin stain. A and B: Anti-Rab38 antibody. C and D: Control (antibody plus synthetic peptides). Original magnification: ×400 (A and C). ×1,000 (B and D). b: Immunohistochemisty of native Rab38 protein for rat bronchial tissue (original magnification, ×1,000). Experimental procedures are same as described in a. A: Anti-Rab38 antibody. B: Control (antibody plus synthetic peptides).

Figure 5.

In situ hybridization of rab38 in rat lung tissue. Perfused and excised rat lungs were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5- to 7-μm-thick slices. The sections were hybridized with digoxigenin-labeled cRNA probes and then treated with RNase. The sections were treated with sheep anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. Color development was performed in 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt. A: Antisense RNA probe (original magnification, ×40). Some of the positive airway cells are indicated by arrowheads. B: Sense RNA probe (original magnification, ×40). Insets are original magnification of ×200 of the boxed areas. C: Antisense RNA probe (original magnification, ×200). Positive alveolar corner cells are indicated by arrows. D: Sense RNA probe (original magnification, ×200).

Figure 6.

Localization of Rab38 protein in subcellular organelle fractions from freshly isolated alveolar type II cells. Isolated alveolar type II cells were disrupted with a Dounce glass homogenizer. The cell homogenate was divided into two parts. The first was centrifuged with a series of differential centrifugal forces (A). The second portion of homogenate was adjusted to a 0.9 mol/L sucrose solution and loaded on a discontinuous sucrose gradient and centrifuged at 100,000 × g. Each sucrose gradient was grouped into fractions, diluted, and centrifuged (B). Equal 50-μg proteins were loaded on SDS-PAGE, and Western blotting was performed as described in Figure 2 ▶ . A: Lane 1, Total alveolar type II cells; lane 2, nucleus; lane 3, heavy vesicles (15,000 × g, 10 minutes); lane 4, light vesicles (100,000 × g, 3 hours); lane 5, cytosol. B: Lane 1, Total alveolar type II cells; lane 2, 1.48 mol/L sucrose; lane 3, 0.9 mol/L sucrose; lane 4, 0.7 to 0.8 mol/L sucrose; lane 5, 0.4 to 0.6 mol/L sucrose. Native Rab38 is indicated by arrows. Note that 0.4 to 0.6 mol/L sucrose (lane 5) is a lamellar body-enriched fraction.

Figure 7.

a: Double-fluorescence immunocytochemistry by confocal laser-scanning microscopy on isolated rat alveolar type II cells. Adherent cells were fixed and reacted with a rabbit anti-Rab38 polyclonal antibody and a mouse anti-subcellular organelle protein. Second antibodies were conjugated with Alexa 488 fluorophore (green) and Alexa 594 (red). Left: Images are stained with Rab38 with green fluorescence (Alexa 488). Middle: Images are stained of subcellular organelle marker proteins (Alexa 594). Right: Merged images of left and middle. General distribution pattern of Rab38 resembles that of ER. Middle: Images are BiP/GRP78 (B, ER resident protein), GM130 (E, Golgi matrix protein), and TGN38 (H, trans-Golgi network resident protein). b: Double-fluorescence immunocytochemistry by confocal laser-scanning microscopy on isolated rat alveolar type II cells. Experimental procedures are identical to those described in a. Middle: Images are EEA1 (B, early endosome resident protein) and Lamp-1 (E, lysosome-associated transmembrane protein-1). Rab38 seems to show minor co-localization with EEA1. c: Double-fluorescence immunocytochemistry by confocal laser scanning microscopy on isolated rat alveolar type II cells. Experimental procedures are identical to those described in a. Middle: Images are SP-A (B) and SP-B (E). Rab38 seems to show partial co-localization with SP-B.

Figure 7B.