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Cell-type-specific and selectively induced expression of members of the p24 family of putative cargo receptors

Jutta Rötter, Roland P. Kuiper, Gerrit Bouw and Gerard J. M. Martens*

Department of Molecular Animal Physiology, Nijmegen Center for Molecular Life Sciences (NCMLS), University of Nijmegen, Geert Grooteplein Zuid 28, 6525 GA Nijmegen, The Netherlands



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  		Fig. 1. Xenopus p24 proteins and their relationship with p24 proteins from other species. (A) Alignment of members of the p24 protein family in X. laevis. Aligned are the amino acid sequences deduced from cDNA clones, the EST database entry BF611875 (representing Xp24{alpha}2) and the two p24{delta} subfamily members identified previously (Kuiper et al., 2000Go). The cDNAs of the Xenopus p24 proteins have been isolated from a neurointermediate pituitary cDNA library, except for those of Xp24{alpha}2 and -{gamma}1 (isolated from an embryo library). Amino acids that are conserved in at least five sequences are in black boxes. The putative signal peptidase cleavage sites of the N-terminal signal sequences (incomplete for Xp24{alpha}2) are indicated by an arrow. Asterisks indicate the two conserved cysteine residues present in the lumenal domains of all p24 proteins. The predicted transmembrane region (TM) is underlined. (B) Phylogenetic tree of the p24 proteins from mouse (m), Xenopus (X), Drosophila melanogaster (d), Caenorhabditis elegans (c) and p24{alpha}1 from dog, and the classification in the four proposed p24 subfamilies. (C) Amino acid sequence identity (%) between p24 proteins of mouse and X. laevis. For sequence comparisons and phylogenetic tree construction, the p24 proteins without their signal sequences were aligned by the Clustal W algorithm using default parameters (AlignX program in Vector NTI Suite 6; InforMax, North Bethesda, MD). The mp24{alpha}1 protein lacks part of the N-terminal region (indicated with an asterisk). Sequences are mostly compilations of several data base entries and accession numbers are available upon request.

 


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  		Fig. 2. p24 protein expression in Xenopus pituitary. The neurointermediate lobe (NIL) was manually dissected from the anterior lobe (AL) of the pituitary of black- or white-adapted Xenopus. 20 µg of NIL and AL extracts were resolved together with tissue extracts from brain and hypothalamus (~60 µg each) by SDS-PAGE. For immunoblotting, antisera directed against sequences in the lumenal domains of the respective p24 proteins or the anti-{delta}C antiserum recognizing Xp24{delta}1 and -{delta}2 were used. The asterisk indicates an AL protein band presumably resulting from crossreactivity of the anti-Xp24{gamma}1 antiserum with an abundant 23 kDa protein (likely growth hormone/prolactin).

 


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  		Fig. 3. Immunocytochemical analysis visualizing the expression of p24 proteins in the pituitary gland of Xenopus laevis adapted to a black or white background. Shown are sagital sections stained with the peroxidase-anti-peroxidase method for Xp24{alpha}3 (affinity-purified anti-{alpha}3L, 1:300 dilution), Xp24ß1 (anti-ß1L, 1:200 dilution), Xp24{gamma}1 (anti-{gamma}1L, 1:600 dilution), Xp24{gamma}2 (affinity-purified anti-{gamma}2L, 1:40 dilution), Xp24{gamma}3L (anti-{gamma}3L, 1:200 dilution), Xp24{delta}1 (affinity-purified anti-{delta}1, 1:50 dilution), and Xp24{delta}2 (anti-{delta}2, 1:1500 dilution). NL, neural lobe; IL, intermediate lobe; AL, anterior lobe. Bar, 200 µm.

 


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  		Fig. 4. p24 expression in Xenopus tissues. (A) Nothern blot analysis of Xenopus p24{alpha}3, -ß1, and -{gamma}3 mRNAs. Equal amounts of RNA were loaded and Xenopus GAPDH was used as a control for RNA loading and integrity. The positions of the 18S and 28S Xenopus ribosomal RNAs are indicated. (B) Western blot analysis of the Xenopus p24 proteins using antibodies against lumenal epitopes of the respective p24 proteins.

 

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