The C terminus of annexin II mediates binding to F-actin

Annexin II heterotetramer (AIIt) is a multifunctional Ca(2+)-binding protein composed of two 11-kDa subunits and two annexin II subunits. The annexin II subunit contains the binding sites for anionic phospholipids, heparin, and F-actin, whereas the p11 subunit provides a regulatory function. The F-actin-binding site is presently unknown. In the present study we have utilized site-directed mutagenesis to create annexin II mutants with truncations in the C terminus of the molecule. Interestingly, a mutant annexin II lacking its C-terminal 16, 13, or 9 amino acids was unable to bind to F-actin but still retained its ability to interact with both anionic phospholipids and heparin. Recombinant AIIt, composed of wild-type p11 subunits and the mutant annexin II subunits, was also unable to bundle F-actin. This loss of F-actin bundling activity was directly attributable to the inability of mutant AIIt to bind F-actin. These results establish for the first time that the annexin II C-terminal amino acid residues, LLYLCGGDD, participate in F-actin binding.     

Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43

Phosphorylation and lipidation provide posttranslational mechanisms that contribute to the distribution of cytosolic proteins in growing nerve cells. The growth-associated protein GAP43 is susceptible to both phosphorylation and S-palmitoylation and is enriched in the tips of extending neurites. However, how phosphorylation and lipidation interplay to mediate sorting of GAP43 is unclear. Using a combination of biochemical, genetic, and imaging approaches, we show that palmitoylation is required for membrane association and that phosphorylation at Ser-41 directs palmitoylated GAP43 to the plasma membrane. Plasma membrane association decreased the diffusion constant fourfold in neuritic shafts. Sorting to the neuritic tip required palmitoylation and active transport and was increased by phosphorylation-mediated plasma membrane interaction. Vesicle tracking revealed transient association of a fraction of GAP43 with exocytic vesicles and motion at a fast axonal transport rate. Simulations confirmed that a combination of diffusion, dynamic plasma membrane interaction and active transport of a small fraction of GAP43 suffices for efficient sorting to growth cones. Our data demonstrate a complex interplay between phosphorylation and lipidation in mediating the localization of GAP43 in neuronal cells. Palmitoylation tags GAP43 for global sorting by piggybacking on exocytic vesicles, whereas phosphorylation locally regulates protein mobility and plasma membrane targeting of palmitoylated GAP43.     

Binding relationships of membrane tethering components. The giantin N terminus and the GM130 N terminus compete for binding to the p115 C terminus

By forming a molecular tether between two membranes, p115, giantin, and GM130 may mediate multiple Golgi-related processes including vesicle transport, cisternae formation, and cisternal stacking. The tether is proposed to involve the simultaneous binding of p115 to giantin on one membrane and to GM130 on another membrane. To explore this model, we tested for the presence of the putative giantin-p115-GM130 ternary complex. We first mapped p115-binding site in giantin to a 70-amino acid coiled-coil domain at the extreme N terminus, a position that may exist up to 400 nm away from the Golgi membrane. We then generated glutathione S-transferase (GST) fusion proteins containing either giantin's or GM130's p115 binding site and tested whether such proteins could bind p115 and GM130 or bind p115 and giantin, respectively. Unexpectedly, GST fusions containing either the giantin or the GM130 p115 binding site efficiently bound p115, but the p115 bound to GST-giantin did not bind GM130, and the p115 bound to GST-GM130 did not bind giantin. To explain this result, we mapped the giantin binding site in p115 and found that it is located at the C-terminal acidic domain, the same domain involved in binding GM130. The presence of a single binding site in p115 for giantin and GM130 was confirmed by demonstration that giantin and GM130 compete for binding to p115. These results question a simple tethering model involving a ternary giantin-p115-GM130 complex and suggest that p115-giantin and p115-GM130 interactions might mediate independent membrane tethering events.     

N terminus of calpain 1 is a mitochondrial targeting sequence

The ubiquitous m- and mu-calpains are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. Previously, mu-calpain was found to be enriched in mitochondrial fractions isolated from rat cerebral cortex and SH-SY5Y neuroblastoma cells, but the submitochondrial localization of mu-calpain was not determined. In the present study, submitochondrial fractionation and digitonin permeabilization studies indicated that both calpain 1 and calpain small subunit 1, which together form mu-calpain, are present in the mitochondrial intermembrane space. The N terminus of calpain 1 contains an amphipathic alpha-helical domain, and is distinct from the N terminus of calpain 2. Calpain 1, but not calpain 2, was imported into mitochondria. Removal of the N-terminal 22 amino acids of calpain 1 blocked the mitochondrial calpain import, while addition of this N-terminal region to calpain 2 or green fluorescent protein enabled mitochondrial import. The N terminus of calpain 1 was not processed following mitochondrial import, but was removed by autolysis following calpain activation. Calpain small subunit 1 was not directly imported into mitochondria, but was imported in the presence of calpain 1. The presence of a mitochondrial targeting sequence in the N-terminal region of calpain 1 is consistent with the localization of mu-calpain to the mitochondrial intermembrane space and provides new insight into the possible functions of this cysteine protease.     

Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane

The exocyst is an octameric protein complex implicated in the tethering of post-Golgi secretory vesicles to the plasma membrane before fusion. The function of individual exocyst components and the mechanism by which this tethering complex is targeted to sites of secretion are not clear. In this study, we report that the exocyst subunit Exo70 functions in concert with Sec3 to anchor the exocyst to the plasma membrane. We found that the C-terminal Domain D of Exo70 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues on Exo70 that are critical for its interaction with phospholipids and the small GTPase Rho3. Further genetic and cell biological analyses suggest that the interaction of Exo70 with phospholipids, but not Rho3, is essential for the membrane association of the exocyst complex. We propose that Exo70 mediates the assembly of the exocyst complex at the plasma membrane, which is a crucial step in the tethering of post-Golgi secretory vesicles for exocytosis.     

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.     

The dimerization of ferrihaems. II. Equilibrium and kinetic studies of mesoferrihaem dimerization.

Spectrophotometric data have been determined for mesoferrihaem at several pH values and over a range of concentration covering four orders of magnitude. The data reveal a dimerization process according to the equation 2 monomer in equilibrium dimer + H+, analogous to earlier findings for deuteroferrihaem and protoferrihaem. The value of K (defined as K = [dimer] [H+]/[monomer]2) was found to be 6.92.10(-2). This is close to the value for deuteroferrihaem but much less than that for protoferrihaem. This is interpreted in terms of possible additional bonding between the delocalized electron systems in protoferrihaem dimers relative to those of mesoferrihaem and deuteroferrihaem. Rate constants for dimerization were determined by temperature-jump spectrophotometry. The pH dependence of the rate constants is explained in terms of two distinct pathways for the dimerization process. These involve either direct reaction between two undissociated monomer molecules or alternatively an initial acid dissociation of a monomer molecule followed by reaction between an undissociated and dissociated molecule.     

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 dimerization of ferrihaems: II. Equilibrium and kinetic studies of mesoferrihaem dimerization

Spectrophotometric data have been determined for mesoferrihaem at several pH values and over a range of concentration covering four orders of magnitude. The data reveal a dimerization process according to the equation 2 monomer ? dimer + H⁺, analogous to earlier findings for deuteroferrihaem and protoferrihaem. The value of K (defined as $\text{K}\text{=}\text{[dimer][H}{}^{\mathrm{+}}\text{]}\text{[}\text{monomer}\text{])}{}^2\text{)}$ was found to be 6.92 · 10^?2. This is close to the value for deuteroferrihaem but much less than that for protoferrihaem. This is interpreted in terms of possible additional bonding between the delocalized electron systems in protoferrihaem dimers relative to those of mesoferrihaem and deuteroferrihaem.Rate constants for dimerization were determined by temperature-jump spectrophotometry. The pH dependence of the rate constants is explained in terms of two distinct pathways for the dimerization process. These involve either direct reaction between two undissociated monomer molecules or alternatively an initial acid dissociation of a monomer molecule followed by reaction between an undissociated and a dissociated molecule.     

Carboxy terminus of glucose transporter 3 contains an apical membrane targeting domain

We previously demonstrated that distinct facilitative glucose transporter isoforms display differential sorting in polarized epithelial cells. In Madin-Darby canine kidney (MDCK) cells, glucose transporter 1 and 2 (GLUT1 and GLUT2) are localized to the basolateral cell surface whereas GLUTs 3 and 5 are targeted to the apical membrane. To explore the molecular mechanisms underlying this asymmetric distribution, we analyzed the targeting of chimeric glucose transporter proteins in MDCK cells. Replacement of the carboxy-terminal cytosolic tail of GLUT1, GLUT2, or GLUT4 with that from GLUT3 resulted in apical targeting. Conversely, a GLUT3 chimera containing the cytosolic carboxy terminus of GLUT2 was sorted to the basolateral membrane. These findings are not attributable to the presence of a basolateral signal in the tails of GLUTs 1, 2, and 4 because the basolateral targeting of GLUT1 was retained in a GLUT1 chimera containing the carboxy terminus of GLUT5. In addition, we were unable to demonstrate the presence of an autonomous basolateral sorting signal in the GLUT1 tail using the low-density lipoprotein receptor as a reporter. By examining the targeting of a series of more defined GLUT1/3 chimeras, we found evidence of an apical targeting signal involving residues 473-484 (DRSGKDGVMEMN) in the carboxy tail. We conclude that the targeting of GLUT3 to the apical cell surface in MDCK cells is regulated by a unique cytosolic sorting motif.     

The amino terminus of the herpes simplex virus 1 protein Vhs mediates membrane association and tegument incorporation

Assembly of herpes simplex viruses (HSV) is a poorly understood process involving multiple redundant interactions between large number of tegument and envelope proteins. We have previously shown (G. E. Lee, G. A. Church, and D. W. Wilson, J. Virol. 77:2038-2045, 2003) that the virion host shutoff (Vhs) tegument protein is largely insoluble in HSV-infected cells and is also stably associated with membranes. Here we demonstrate that both insolubility and stable membrane binding are stimulated during the course of an HSV infection. Furthermore, we have found that the amino-terminal 42 residues of Vhs are sufficient to mediate membrane association and tegument incorporation when fused to a green fluorescent protein (GFP) reporter. Particle incorporation correlates with sorting to cytoplasmic punctate structures that may correspond to sites of HSV assembly. We conclude that the amino terminus of Vhs mediates targeting to sites of HSV assembly and to the viral tegument.     

The syntaxin 4 N terminus regulates its basolateral targeting by munc18c-dependent and -independent mechanisms

To generate and maintain epithelial cell polarity, specific sorting of proteins into vesicles destined for the apical and basolateral domain is required. Syntaxin 3 and 4 are apical and basolateral SNARE proteins important for the specificity of vesicle fusion at the apical and basolateral plasma membrane domains, respectively, but how these proteins are specifically targeted to these domains themselves is unclear. Munc18/SM proteins are potential regulators of this process. Like syntaxins, they are crucial for exocytosis and vesicle fusion. However, how munc18c and syntaxin 4 regulate the function of each other is unclear. Here, we investigated the requirement of syntaxin 4 in the delivery of basolateral membrane and secretory proteins, the basolateral targeting of syntaxin 4, and the role of munc18c in this targeting. Depletion of syntaxin 4 resulted in significant reduction of basolateral targeting, suggesting no compensation by other syntaxin forms. Mutational analysis identified amino acids Leu-25 and to a lesser extent Val-26 as essential for correct localization of syntaxin 4. Recently, it was shown that the N-terminal peptide of syntaxin 4 is involved in binding to munc18c. A mutation in this region that affects munc18c binding shows that munc18c binding is required for stabilization of syntaxin 4 at the plasma membrane but not for its correct targeting. We conclude that the N terminus serves two functions in membrane targeting. First, it harbors the sorting motif, which targets syntaxin 4 basolaterally in a munc18c-independent manner and second, it allows for munc18c binding, which stabilizes the protein in a munc18c-dependent manner.     

Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells

The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P(2). Disrupting Exo70-PI(4,5)P(2) interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P(2)-binding-deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P(2) interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.     

The N-terminus of Drosophila ESC mediates its phosphorylation and dimerization

The ESC protein, like other Polycomb Group proteins, is required for heritable silencing of the homeotic genes. ESC is phosphorylated in vivo, but the region of ESC that is phosphorylated and its consequences are not known. Here, we show that the amino-terminal region of ESC (residues 1-60) mediates its phosphorylation and dimerization. Phosphorylation of ESC1-60 in vitro by CK1 and CK2 strongly enhances its dimerization. Both phosphorylation and dimerization are conserved in the mammalian ESC homolog EED, suggesting that they play important roles in vivo. One role is suggested by the effect of phosphatase treatment on native ESC complexes, which does not affect the integrity of the 600 kDa ESC/E(Z) complex, but eliminates the 1 MDa ESC/E(Z) complex, which is distinguished from the former by the presence of the additional subunits PCL and RPD3. Thus, stability and perhaps assembly of larger ESC complexes may depend on ESC phosphorylation.     

The calmodulin-binding domain mediates the self-association of the plasma membrane Ca2+ pump.

Dimerization (oligomerization) of the plasma membrane Ca2+ pump increases its activity (Kosk-Kosicka, D., Bzdega, T., and Wawrzynow, A. (1989) J. Biol. Chem. 264, 19495-19499). Fluorescence titration on preparations of the purified eosin-labeled human erythrocyte ATPase has been used to monitor the oligomerization process. Calmodulin inhibits oligomerization, although it can bind to the oligomerized enzyme. Synthetic peptides corresponding to the calmodulin-binding domain of the pump stimulate its ATPase activity, indicating the formation of heterooligomers of the peptides with the pump. The oligomerization is prevented by the preincubation of the ATPase with calmodulin. Polyclonal antibodies against the synthetic calmodulin-binding domain inhibit its basal and its calmodulin-stimulated ATPase activity and prevent the formation of the oligomers. ATPase preparations truncated at the COOH terminus with calpain to a fragment of 124 kDa which does not contain the calmodulin-binding domain fail to oligomerize with the intact ATPase. The results show that the calmodulin-binding domain mediates the oligomerization of the Ca2+ pump.     

The lumenal domain of the integral membrane protein phogrin mediates targeting to secretory granules

Phogrin, a transmembrane glycoprotein of neuroendocrine cells, is localized to dense-core secretory granules. We have investigated the subcellular targeting of phogrin by analyzing the sorting of a series of deletion mutants to the regulated pathway of secretion in AtT20 cells. The lumenal domain as a soluble protein was efficiently routed to granules, based on a combination of morphological analysis and secretion studies. Sorting was not dependent on a candidate targeting signal consisting of an N-terminal conserved cysteine-rich motif. Both the pro-region and the lumenal domain of mature, post-translationally processed phogrin independently reached the granule, although the pro-region was sorted more efficiently. Once within the regulated secretory pathway, all phogrin lumenal domain proteins were stored in functional granules for extended periods of time. Thus, phogrin possesses several domains contributing to its targeting to the secretory granule. Our findings support a model of granule biogenesis where proteins are sorted on the basis of their biochemical properties rather than via signal-dependent binding to a targeting receptor. Sorting of integral membrane proteins mediated by the lumenal domain may ensure that functionally important transmembrane molecules are included in the forming granule.     

The targeting of the plasma membrane calcium pump in the cell

The information on the structural determinants that control the cellular distribution of P-type pumps is very scarce. However, recent experiments on the membrane targeting of the plasma membrane Ca²⁺ pump (PMCA) have provided interesting leads on the problem: they will be discussed in this succinct review. A general introduction on the biochemical properties of the PMCA pump will preface the discussion of the specific findings on the role of three distinct regions of the molecule in the targeting process.     

The membrane proteins of the vacuolar system. II. Bidirectional flow between secondary lysosomes and plasma membrane

Lactoperoxidase covalently coupled to latex spheres (LPO-latex) has been used to selectively iodinate the phagolysome (PL) membrane within living macrophages, as discussed in the accompanying article. This procedure labeled approximately 24 polypeptides in the PL membrane; these were similar to those iodinatable on the external surface of the plasma membrane (PM). We now report on the translocation and fate of these proteins when the cells are returned to culture. TCA-precipitable radioactivity was lost from cells with biphasic kinetics. 20-50% of the cell-associated radiolabel was rapidly digested (t 1/2 approximately equal to 1 h) and recovered in the culture medium as monoiodotyrosine. 50-80% of the label was lost slowly from cells ( 1/2 approximately equal to 24-30 h). Quantitative analysis of gel autoradiograms showed that all radiolabeled proteins were lost at the same rate in both the rapid and slow phases of digestion. Within 15-30 min aftr labeling of the PL membrane, EM autoradiography revealed that the majority of the cell-associated grains, which at time 0 were associated with PL, were now randomly dispersed over the plasmalemma. At this time, analysis of PM captured by a second phagocytic load revealed the presence of all labeled species originally present in the PL membrane. This demonstrated the rapid, synchronous centrifugal flow of PL polypeptides to the cell surface. Evidence was also obtained for the continuous influx of representative samples of the PM into the PL compartment by way of pinocytic vesicles. This was based on the constant flow of fluid phase markers into latex-containing PL and on the internalization of all iodinatable PM polypeptides into this locus. These observations provide evidence for the continuous, bidirectional flow of membrane polypeptides between the PM and the secondary lysosome and represent an example of a membrane flow and recycling mechanism.