A tightly membrane-associated subpopulation of spectrin is 3H-palmitoylated

Laboratory for Biochemistry, Swiss Federal Institute of Technology, ETH-Zentrum, Zurich.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/1993; 268(17):12996-3001.
Source: PubMed

ABSTRACT A tightly membrane-associated form of spectrin (TMA-spectrin) was labeled when human red blood cells were incubated with [3H]palmitic acid. About 90% of spectrin was not fatty acid-acylated and was extracted from membranes by low salt buffers. The 3H-palmitoylated TMA-spectrin, however, resisted low and even high salt extraction and remained associated with inside-out vesicles that were generated in the process of spectrin-actin extraction from membranes. TMA-spectrin was preferentially extracted from KCl-stripped vesicles by 5 M urea at low ionic strength. TMA-spectrin was purified by gel filtration and by ion exchange chromatography in the presence of urea and a non-ionic detergent. Purified TMA-spectrin was 3H-palmitoylated exclusively in the beta subunit to 0.28 mol/mol after a 12-h incubation of red cells. The labeled palmitate may be bound as an ester or thioester, since hydroxylamine (1 M, pH 7.5) released the label completely. Peptide maps of 3H-palmitoylated TMA-spectrin showed three or two labeled peptides from the beta subunit, when generated by V8 protease and trypsin, respectively. Two types of antibodies to spectrin reacted with purified TMA-spectrin, and TMA-spectrin contained the same antigenic peptides as low salt-extractable spectrin. Rabbit anti-ankyrin antibodies did not bind to TMA-spectrin. The substoichiometric incorporation of [3H]palmitic acid into TMA-spectrin could result from the slow turnover of endogenously bound fatty acids. Generation of the tightly membrane-associated and 3H-palmitoylated subpopulation of spectrin cannot be due to entrapment of an unmodified residual fraction of spectrin in right-side-out vesicles. Instead, the data suggest the existence of a subpopulation of spectrin molecules that undergo a covalent fatty acid modification and thereby alter their binding properties. This may offer a new, metabolically dependent mechanism for dynamic interactions between spectrin and the membrane lipid bilayer.

Download full-text


Available from: Hans Ulrich Lutz, Jul 30, 2015
  • Source
    • "Although the fast kinetics of carbonate action (30 min at 4 °C, Fig. 1C) would argue in favour of this view, other mechanisms cannot be ruled out. A study is under way to evaluate the possible contribution of S-acylated (palmitoylated) proteins in the membrane skeleton [41] [42], such as p55, spectrin and ankyrin, for the anchoring of DRMs, through the insertion of their saturated palmitoyl groups into the liquid-ordered phase of these microdomains: the alkalinization brought about by carbonate might be sufficient for hydrolysing these relatively weak thioester bonds. We previously reported that the integral proteins band 3 and GPC were present at low, but significant levels in DRMs, and speculated that they could be responsible for the anchoring of DRMs to the membrane skeleton [15]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Lipid rafts are local inhomogeneities in the composition of the plasma membrane of living cells, that are enriched in sphingolipids and cholesterol in a liquid-ordered state, and proteins involved in receptor-mediated signalling. Interactions between lipid rafts and the cytoskeleton have been observed in various cell types. They are isolated as a fraction of the plasma membrane that resists solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). We have previously described that DRMs are anchored to the spectrin-based membrane skeleton in human erythrocytes and can be released by increasing the pH and ionic strength of the solubilization medium with sodium carbonate. It was unexplained why this carbonate treatment was necessary and why this requirement was not reported by other workers in this area. We show here that when contaminating leukocytes are present in erythrocyte preparations that are subjected to detergent treatment, the isolation of DRMs can occur without the requirement for carbonate treatment. This is due to the uncontrolled breakdown of erythrocyte membrane components by hydrolases that are released from contaminating neutrophils that lead to proteolytic disruption of the supramolecular assembly of the membrane skeleton. Results presented here corroborate the concept that DRMs are anchored to the membrane skeleton through electrostatic interactions that most likely involve the spectrin molecule.
    Biochimica et Biophysica Acta 01/2011; 1808(1):183-90. DOI:10.1016/j.bbamem.2010.08.019 · 4.66 Impact Factor
  • Source
    • "However, despite their localization in association with membranes, none of the conventional calpains is acylated. It is interesting to note, however, that some of the established in vivo substrates of mammalian calpains, the cytoskeletal proteins vinculin , spectrin, ankyrin, and band 4.1, are themselves acylated and membrane-associated (Staufenbiel and Lazarides 1986; Burn and Burger 1987; Maretzki et al. 1990; Mariani et al. 1993; Bhatt et al. 2002). An additional group of calpain-like proteins with its own distinctive feature are seven sequences that are characterized by the presence of internal repeats of 65–68 amino acids that are homologous between all seven sequences. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Employing whole-genome analysis we have characterized a large family of genes coding for calpain-related proteins in three kinetoplastid parasites. We have defined a total of 18 calpain-like sequences in Trypanosoma brucei, 27 in Leishmania major, and 24 in Trypanosoma cruzi. Sequence characterization revealed a well-conserved protease domain in most proteins, although residues critical for catalytic activity were frequently altered. Many of the proteins contain a novel N-terminal sequence motif unique to kinetoplastids. Furthermore, 24 of the sequences contain N-terminal fatty acid acylation motifs indicating association of these proteins with intracellular membranes. This extended family of proteins also includes a group of sequences that completely lack a protease domain but is specifically related to other kinetoplastid calpain-related proteins by a highly conserved N-terminal domain and by genomic organization. All sequences lack the C-terminal calmodulin-related calcium-binding domain typical of most mammalian calpains. Our analysis emphasizes the highly modular structure of calpains and calpain-like proteins, suggesting that they are involved in diverse cellular functions. The discovery of this surprisingly large family of calpain-like proteins in lower eukaryotes that combines novel and conserved sequence modules contributes to our understanding of the evolution of this abundant protein family.
    Journal of Molecular Evolution 01/2006; 61(6):742-57. DOI:10.1007/s00239-004-0272-8 · 1.86 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Recent advances have been made in our understanding of the direct binding of actin to integral membrane proteins. New information has been obtained about indirect actin-membrane associations through spectrin superfamily members and through proteins at the cytoplasmic surfaces of focal contacts and adherens junctions.
    Current Opinion in Cell Biology 03/1994; 6(1):120-30. DOI:10.1016/0955-0674(94)90125-2 · 8.74 Impact Factor
Show more