In the plasma membrane, spectrin family members are known to function in facilitating membrane association with the cytoskeleton, the promotion of general membrane stability, and the formation of discrete membrane domains

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In the plasma membrane, spectrin family members are known to function in facilitating membrane association with the cytoskeleton, the promotion of general membrane stability, and the formation of discrete membrane domains. binding domains of standard spectrins. Golgi localization for this spectrin-like protein was shown by manifestation of epitope-tagged fragments in MDBK and COS cells, identifying two unique Golgi binding sites, and by immunofluorescence microscopy by using several different antibody preparations. One of the Golgi binding domains on Syne-1 functions as a dominating bad inhibitor that alters the structure of the Golgi complex, which collapses into a condensed structure near the centrosome in transfected epithelial A-485 cells. We conclude the Syne-1 gene is definitely expressed in a variety of forms that are multifunctional and are capable of functioning at both the Golgi and the nuclear envelope, maybe linking the two organelles during muscle mass differentiation. Intro The spectrin superfamily is definitely comprised of a variety of functionally and structurally related cytoskeletal proteins that include several of isoforms of spectrin (Bennett, 1990 ), dystrophin A-485 (Koenig strain XL1-blue. Peptide antibodies were prepared against the following peptides: EAKASSPEMDISADC (SN357-1 and SN357-2) and EESGEEGTNSEISSC (SN119 and SN120). Peptide synthesis, conjugation to KLH, and antibody production was performed at SynPep (Dublin, CA). Ectopic Manifestation of Syne-1 Fragments For ectopic manifestation of various regions of Syne-1, epitope-tagged constructs were prepared using the identical strategy explained above for building of GST fusion proteins. PCR products digested with -Spectrin (11)a 100 (100) 17 (37) -Spectrin (21)b 55 (72) 17 (37) -Actinin 31 (53) 20 (39) -Spectrin 26 (47) 17 (38) Golgi -spectrin 25 (44) A-485 17 (37) Dystrophin 17 (39) 17 (39) MDBK clone 4 17 (37) 100 (100) Syne-1 17 (37) 85 (90) Open in a separate window Optimal, local sequence alignments were performed with the complete amino acid sequences of the indicated spectrin superfamily users by using BLAST 2.0 (National Center for Biotechnology Info). Sequences are compared with erythroid -spectrin because the -subunit of spectrin lacks an actin binding site, therefore any bias in overall homology due to the presence highly conserved actin binding site (not present in MDBK clone 4) is definitely avoided. All sequences except CCL4 MDBK clone 4 are from human being clones aerythroid -spectrin bNonerythroid -spectrin Syne-1/Nesprin-1 has been show to be indicated in multiple forms (Apel Golgi/intermediate compartment marker p58 (Number 4j). In addition to the Golgi localized transmission, we also observed diffuse cytoplasmic localization with these fragments, consistent with a cytoplasmic pool of protein in equilibrium having a Golgi bound fraction. Somewhat less obvious Golgi localization was observed with the HAf2 fragment (Number 4a) than with HAf3. Typically, Golgi-specific staining was more difficult to detect with HAf2 fragment due to higher cytoplasmic staining compared with that observed with HAf3, suggesting the HAf2 A-485 fragment binds Golgi with a lower affinity. Golgi localization was not observed with HAf4, which offered a diffuse cytoplasmic transmission, or HAf1, which localized to discrete cytoplasmic puncta that did not correspond to any known organelle (our unpublished data). These results demonstrate a capacity for Syne-1 to bind and localize to the Golgi complex. Furthermore, they also determine at least two unique Golgi binding sites on Syne-1, a low-affinity site in the vicinity of amino acids 4606C4945, and a high-affinity site within amino acids 5015C5410. It should be noted that we did not notice nuclear envelope localization with any of the fragments tested. Open in a separate window Number 4. Ectopically indicated fragments of Syne-1 determine Golgi binding determinants. To demonstrate Golgi localization for.