Abstract
We report here on the isolation, cloning, and expression of two Mr 21,000 proteins from rat pancreatic acinar cells, the rat-Tmp21 (transmembrane protein, Mr 21,000) and the rat-p24A. Both proteins are transmembrane proteins with type I topology and share weak but significant homology to one another (23% identity). We further show the cloning and characterization of the human homologs, hum-Tmp21, which is expressed in two variants (Tmp21-I and Tmp21-II), and hum-p24A. Tmp21 proteins and p24A have highly conserved COOH-terminal tails, which contain motifs related to the endoplasmic reticulum retention and retrieval consensus sequence KKXX. The rat-p24 sequence is identical to the hamster CHOp24, a recently characterized component of coatomer-coated transport vesicles, which defines a family of proteins (called the p24 family) proposed to be involved in vesicular transport processes (Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P., and Rothman, J. E.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8011-8015). Sequence alignment and structural features identify the Tmp21 protein as a new member of this p24 family. Northern analysis of various tissues indicates that the Tmp21 proteins and the p24A protein are ubiquitously expressed. The integral membrane components Tmp21 and p24A are localized in microsomal membranes, zymogen granule membranes, and the plasma membrane and are absent from the cytosol. Both p24A and Tmp21 show weak homology to the yeast protein Emp24p, which recently has been shown to be involved in secretory protein transport from the endoplasmic reticulum to the Golgi apparatus. This leads us to conclude that the receptor-like Tmp21 and p24A are involved in vesicular targeting and protein transport.
Highlights
The current knowledge of vesicular transport mechanisms concerns mainly the budding, docking, and fusion of transport vesicles
All peptide sequences identified by microsequencing of the Mr 21,000/22,000 protein bands were encoded within the identified open reading frames (ORF) of the ratTmp21-I and rat-p24A cDNA screening clones
Topology of the Tmp21 and p24A Proteins in Microsomal Membranes—Hydropathy analysis of Tmp21 and p24A revealed a stretch of about 21 amino acids close to the COOH terminus that is sufficiently long to span the lipid bilayer. This suggests that Tmp21 and p24A are type I transmembrane proteins with a large luminal domain and a short COOH terminal stretch of about 1 kDa, which is exposed to the cytosol
Summary
Preparation of Pancreatic Acinar Cells and Subcellular Fractions— Acinar cells were isolated from rat pancreas by collagenase digestion (Streb and Schulz, 1983). For Western blot analysis, proteins were electrophoretically separated on SDS-PAGE and transferred to nitrocellulose membranes. Amplification and Cloning of cDNA Probes—Degenerated oligonucleotides encoding Tmp and p24A peptide sequences were synthesized and used for the reverse transcription-polymerase chain reaction. We used cDNA obtained from standard reverse-transcriptase reactions (Sambrook et al, 1989) with RNA, which had been isolated from different human and rat tissues by the method of Chomczynski and Sacchi (1987). The expressed proteins were used to confirm the identity of the cloned Tmp and p24A cDNAs to the microsomal proteins and for affinity purification of the antibodies described above. Samples containing 20 g of protein taken at different time points were analyzed by Western blot analysis using the anti-Tmp and anti-p24A antibodies
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