A widely expressed betaIII spectrin associated with Golgi and cytoplasmic vesicles - PubMed (original) (raw)

A widely expressed betaIII spectrin associated with Golgi and cytoplasmic vesicles

M C Stankewich et al. Proc Natl Acad Sci U S A. 1998.

Abstract

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the beta-spectrin gene family by homology searches of the GenBank databases and by 5' rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called betaIII spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrin-homology domain. betaIII spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human betaIII spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human betaIII spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that betaIII spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, trans-Golgi network (TGN)38, and beta-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with betaIII spectrin. betaIII spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.

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Figures

Figure 1

Structure of βIII spectrin. (A) Relationship of the cDNA clones forming the βIII spectrin contig representing 7,938 nt. Homologous mouse clones are shown in italics. Clones fully sequenced in this work are shaded. After this work was completed, the full-length sequences of human and rat βIII spectrin were published independently in two separate reports (47, 48). (B) The ORF is predicted to encode a protein of 271,294 Da (2,391 amino acids) with potential functional domains similar to other spectrins. The actin-binding domain (ABD, shaded area) and protein 4.1-binding domain (4.1) are near the N terminus. They are followed by 17 tandem spectrin repeats. Repeats 1, 15, and 17 contain a potential membrane-association domain (MAD1), ankyrin-binding domain (ANK), and spectrin self-association site, respectively. Near the C terminus is a second membrane-association domain (MAD2, shaded) and the PH (boxed). The two peptides used to generate βIII spectrin-reactive antibodies are shown by the black bars. A comparison of the amino acid sequences of βIΣ2 and βIIΣ1 spectrins with βIII spectrin reveals extensive homology in the N-terminal domain, the repeats domain, and the PH domain, as shown by the identity-vs.-amino acid number plot. Each point represents the percent identity of 20 aligned amino acids. (C) Chromosome localization of βIII spectrin. (Left) The location of the human βIII spectrin gene (SPTBN2) on the long arm of chromosome 11 (11q13) is shown relative to other markers mapped in the panel. Gene symbols are on the right and genetic distances between markers in centirays are on the left. PC denotes the gene for pyruvate carboxylase. (Right) The location of the mouse βIII spectrin gene (Spnb3) on chromosome 19 is shown relative to other markers. Gene symbols are on the right and genetic distances between markers (centimorgans ± SE) are on the left. Gene symbols are Mtvr2, a mammary tumor virus receptor; Tbx10, a member of the Tbx1-subfamily of developmental genes; Rn18s-rs6, 18S-ribosomal RNA-related sequence 6; Zp1, zona pellucida glycoprotein 1; Lpc1, lipocortin 1; and Pcsk5, proprotein convertase subtilisin/kexin type 5. Detailed references are available at

http://www.jax.org/resources/documents/cmdata/

Figure 2

Northern blots of electrophoretically separated human poly(A)+ mRNAs (≈2 mg). (Left) Adult tissues from various sources (as indicated) hybridized to clone 184038 (Upper Left) to identify βIII spectrin transcripts or hybridized to actin (Lower Left) as a control for loading. Note that actin is overexpressed in heart and skeletal muscle and cannot be used as a control in those tissues. (Right) Similar analysis of human fetal tissues hybridized to a PCR product representing nucleotides 6,445–6,737.

Figure 3

Cellular distribution of βIII spectrin. Indirect immunofluorescence studies of MDCK (A and B) and NRK (C and D) cells stained with (Left) anti-βIII spectrin antibodies (Spb3C2, A, C, and D; PAb R-βIII7–11, B) or with (Center) anti-β-COP (A), anti-Bip (B); anti-ManII (C), or anti-TGN38 (D). Merged images are also shown (Right). Areas marked by rectangles in B were magnified (B, Right) to show the vesicle-like structures stained with anti-βIII-spectrin antibody (in red) or anti-Bip antibody (in green). βIII spectrin is largely distinct from the distribution of Bip, a marker of the ER. In contrast, there is coincident staining of βIII spectrin with Golgi markers (Right in C and D) and variably with the punctate vesicles stained by those markers. Similar results were also observed with HeLa and COS cells (not shown). (Top, Right) Western blot analysis of MDCK cells (lanes 1–4) and NRK cells (lanes 5 and 6) using PAb R-βIII7–11 (lanes 3–6) reveals an immunoreactive band of ≈220 kDa that is distinct from βII spectrin (lane 2, stained with PAb-10D) and that is not present in pre-immune antisera for PAb R-βIII7–11 (lane 1). The βIII spectrin band was evident in both whole-cell lysates (lanes 1–3, 5) and in RIPA buffer extracts (lanes 4 and 6). Additional immunoreactive bands were inconsistently observed at ≈155 and ≈70 kDa. (Bars = 20 μm.)

Figure 4

βIII spectrin cosegregates with Golgi membranes and other vesicle markers. Fresh rat-liver membranes were floated in a sucrose gradient, and fractions were analyzed for the presence of βIII spectrin and other organelle markers by Western blotting (above each gradient) and densitometry. Also shown (Right) is the intracellular distribution of each marker in MDCK cells by indirect immunofluorescence with the antibody used for the blotting the gradient fractions. (A) βIII spectrin measured with 1:5,000 PAb R-βIII7–11. The enzymatic activity of ManII in each fraction was also measured (•) as a marker of Golgi. Morphologically identifiable Golgi membranes sediment between 0.8 and 1.0 M sucrose (bar). About 16% of βIII spectrin sediments in the Golgi fraction, the rest in denser membrane fractions. (B) The distribution of ankyrin AnkG119. (C) The distribution of calnexin, an ER marker. (D) The distribution of β-COP, a marker of Golgi and COPI transport vesicles.

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A widely expressed betaIII spectrin associated with Golgi and cytoplasmic vesicles - PubMed (original) (raw)