Mechanism Of Membrane Targeting Research Articles

Mutational and biochemical analysis of plasma membrane targeting mediated by the farnesylated, polybasic carboxy terminus of K-ras4B.

Mutational analysis and in vitro assays of membrane association have been combined to investigate the mechanism of plasma membrane targeting mediated by the farnesylated, polybasic carboxy-terminal sequence of K-ras4B in mammalian cells. Fluorescence-microscopic localization of chimeric proteins linking the enhanced green fluorescent protein (EGFP) to the K-ras4B carboxy-terminal sequence, or to variant forms of this sequence, reveals that the normal structure of this targeting motif can be greatly altered without compromising plasma membrane-targeting activity so long as an overall strongly polybasic/amphiphilic character is retained. An EGFP/K-ras4B(171-188) chimeric protein was readily abstracted from isolated cell membranes by negatively charged lipid vesicles, and this abstraction was markedly enhanced by the anionic lipid-binding agent neomycin. Our results strongly favor a mechanism in which at the plasma membrane the carboxy-terminal sequence of K-ras4B associates not with a classical specific proteinaceous receptor but rather with nonspecific but highly anionic 'sites' formed at least in part by the membrane lipid bilayer. Our findings also suggest that the recently demonstrated prenylation-dependent trafficking of immature forms of K-ras4B through the endoplasmic reticulum [Choy et al. (1999) Cell 98, 69-80], while required for maturation of the protein, beyond this stage may not be essential to allow the ultimate delivery of the mature protein to the plasma membrane.

The N-Terminal Matrix Domain of HIV-1 Gag Is Sufficient but Not Necessary for Viral Protein U-Mediated Enhancement of Particle Release through a Membrane-Targeting Mechanism

Viral protein U (Vpu) is an 81 amino acid phosphoprotein found in human immunodeficiency virus type 1 (HIV-1)-infected cells. One function of Vpu is to enhance the release of virus particles from the plasma membrane in infected cells. Using subcellular fractionation, we observed that Vpu promotes the targeting of Pr55 Gag to the plasma membrane, the site of viral assembly. Deletions of Pr55, which removed most of the N-terminal matrix domain (p39) or the C-terminal domains of nucleocapsid and p6 (p41), still allowed for virus-like particle production. Moreover, the release of these particles remained Vpu-responsive. The N-terminal matrix (MA) domain of Gag, which contains its membrane-binding domain, is sufficient for Vpu-mediated enhanced release into the supernatant. Furthermore, a MA-GFP fusion protein showed enhanced membrane binding in the presence of Vpu. This demonstrates that Vpu action may be mediated by allowing Gag, specifically the N-terminal matrix domain, to efficiently associate with the plasma membrane. Thus MA appears sufficient but not necessary for Vpu-mediated enhanced particle release.

8] Molecular structure of membrane-targeting calcium sensors in vision: Recoverin and guanylate cyclase-activating protein 2

Publisher Summary An important class of calcium-binding proteins present in retinal photoreceptor cells includes recoverin from mammalian rods, guanylate cyclase-activating proteins (GCAP-1, GCAP-2, and GCAP-3) from mammalian rods and cones, and guanylate cyclase inhibitory protein (GCIP) from frog rods. The recoverin branch of the EF-hand superfamily includes neuronal Ca 2+ sensors such as neurocalcin, frequenin, and hippocalcin. Mass spectrometric analysis of retinal recoverin and the GCAP proteins revealed that they are myristoylated at the amino terminus. GCAP-2 exhibits different membrane-targeting properties from those of recoverin. The myristoylated and unmyristoylated forms of GCAP-2 bind to membranes at low Ca 2+ concentrations ( 50 mM NaCl).This chapter presents a detailed structural analysis of recoverin and GCAP-2. The Ca 2+ -induced structural changes in these proteins are important for elucidating their membrane-targeting mechanisms and for understanding the molecular mechanism of Ca 2+ -sensitive regulation of phototransduction.

SNAP-25 Is Targeted to the Plasma Membrane through a Novel Membrane-binding Domain

SNAP-25, syntaxin, and synaptobrevin are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. Membrane attachment of syntaxin and synaptobrevin is achieved through a C-terminal hydrophobic tail, whereas SNAP-25 association with membranes appears to depend upon palmitoylation of cysteine residues located in the center of the molecule. This process requires an intact secretory pathway and is inhibited by brefeldin A. Here we show that the minimal plasma membrane-targeting domain of SNAP-25 maps to residues 85-120. This sequence is both necessary and sufficient to target a heterologous protein to the plasma membrane. Palmitoylation of this domain is sensitive to brefeldin A, suggesting that it uses the same membrane-targeting mechanism as the full-length protein. As expected, the palmitoylated cysteine cluster is present within this domain, but surprisingly, membrane anchoring requires an additional five-amino acid sequence that is highly conserved among SNAP-25 family members. Significantly, the membrane-targeting module coincides with the protease-sensitive stretch (residues 83-120) that connects the two alpha-helices that SNAP-25 contributes to the four-helix bundle of the synaptic SNARE complex. Our results demonstrate that residues 85-120 of SNAP-25 represent a protein module that is physically and functionally separable from the SNARE complex-forming domains.

Insertion of the 34-kDa Chloroplast Protein Import Component, IAP34, into the Chloroplast Outer Membrane Is Dependent on Its Intrinsic GTP-binding Capacity

IAP34 is a 34-kDa component of the outer membrane complex that mediates the initial stages of protein import into chloroplasts (Seedorf, M., Waegemann, K., and Soll, J. (1995) Plant J. 7, 401-411; Kessler, F., Blobel, G., Patel, H. A., and Schnell, D. J. (1994) Science 266, 1035-1039). We have investigated the targeting and insertion of IAP34 at the outer envelope membrane. The analyses of IAP34 deletion mutants and hybrid proteins (consisting of regions of IAP34 fused to the soluble IgG-binding domain of staphylococcal protein A) suggest that the transmembrane domain and C-terminal tail of IAP34 contain information essential but not sufficient for targeting to the outer membrane. Treatment of chloroplasts with exogenous proteases does not affect IAP34 insertion, indicating that targeting does not require surface-exposed receptors at the envelope. GTP or GDP is required for maximal integration of IAP34 into the outer membrane. The GTP/GDP requirement is attributed to the intrinsic GTP binding activity of IAP34 because GTP/GDP binding-deficient mutants are defective in outer membrane insertion. On the basis of these observations, we propose that IAP34 is targeted to the chloroplast by a C-terminal signal and efficiently integrated into the outer membrane by conformation-induced insertion upon GTP/GDP binding.

Regulation by phosphorylation of reversible association of a myristoylated protein kinase C substrate with the plasma membrane

Protein kinase C (PKC) transduces receptor-mediated signals by phosphorylating membrane-bound substrates which then act as effectors of specific cellular responses. The myristoylated alanine-rich C kinase substrate (MARCKS) is a specific PKC substrate which has been implicated in macrophage activation, neuro-secretion and growth factor-dependent mitogenesis. Myristoylation of MARCKS is required for effective binding to the plasma membrane where it colocalizes with PKC. Here we report that PKC-dependent phosphorylation displaces MARCKS from the membrane and that its subsequent dephosphorylation is accompanied by its reassociation with the membrane. This cycle of phosphorylation-dependent membrane attachment and detachment of a myristoylated protein represents a novel mechanism of reversible membrane targeting. As MARCKS is a calmodulin- and actin-binding protein (ref. 8, and J. Hartwig et al., manuscript submitted), the cycle of membrane attachment/detachment represents a mechanism through which PKC might reversibly regulate actin-membrane interaction.