Erythrocyte membrane vesiculation: Model for the molecular mechanism of protein sorting

Budding and vesiculation of erythrocyte membranes occurs by a process involving an uncoupling of the membrane skeleton from the lipid bilayer. Vesicle formation provides an important means whereby protein sorting and trafficking can occur. To understand the mechanism of sorting at the molecular level, we have developed a micropipette technique to quantify the redistribution of fluorescently labeled erythrocyte membrane components during mechanically induced membrane deformation and vesiculation. Our previous studies indicated that the spectrin-based membrane skeleton deforms elastically, producing a constant density gradient during deformation. Our current studies showed that during vesiculation the skeleton did not fragment but rather retracted to the cell body, resulting in a vesicle completely depleted of skeleton. These local changes in skeletal density regulated the sorting of nonskeletal membrane components. Highly mobile membrane components, phosphatidylethanolamine- and glycosylphosphatidylinositol-linked CD59 with no specific skeletal association were enriched in the vesicle. In contrast, two components with known specific skeletal association, band 3 and glycophorin A, were differentially depleted in vesicles. Increasing the skeletal association of glycophorin A by liganding its extrafacial domain reduced the fraction partitioning to the vesicle. We conclude that this technique of bilayer/skeleton uncoupling provides a means with which to study protein sorting driven by changes in local skeletal density. Moreover, it is the interaction of particular membrane components with the spectrin-based skeleton that determines molecular partitioning during protein sorting.

The N terminus of the Drosophila Numb protein directs membrane association and actin-dependent asymmetric localization

Drosophila Numb is a membrane associated protein of 557 amino acids (aa) that localizes asymmetrically into a cortical crescent in mitotic neural precursor cells and segregates into one of the daughter cells, where it is required for correct cell fate specification. We demonstrate here that asymmetric localization but not membrane localization of Numb in Drosophila embryos is inhibited by latrunculin A, an inhibitor of actin assembly. We also show that deletion of either the first 41 aa or aa 41–118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. Fusion of the first 76 or the first 119 aa of Numb to β-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and β-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization.

Functioning of the Drosophila integral U1/U2 protein Snf independent of U1 and U2 small nuclear ribonucleoprotein particles is revealed by snf+ gene dose effects

Snf, encoded by sans fille, is the Drosophila homolog of mammalian U1A and U2B′′ and is an integral component of U1 and U2 small nuclear ribonucleoprotein particles (snRNPs). Surprisingly, changes in the level of this housekeeping protein can specifically affect autoregulatory activity of the RNA-binding protein Sex-lethal (Sxl) in an action that we infer must be physically separate from Snf’s functioning within snRNPs. Sxl is a master switch gene that controls its own pre-mRNA splicing as well as splicing for subordinate switch genes that regulate sex determination and dosage compensation. Exploiting an unusual new set of mutant Sxl alleles in an in vivo assay, we show that Snf is rate-limiting for Sxl autoregulation when Sxl levels are low. In such situations, increasing either maternal or zygotic snf+ dose enhances the positive autoregulatory activity of Sxl for Sxl somatic pre-mRNA splicing without affecting Sxl activities toward its other RNA targets. In contrast, increasing the dose of genes encoding either the integral U1 snRNP protein U1-70k, or the integral U2 snRNP protein SF3a60, has no effect. Increased snf+ enhances Sxl autoregulation even when U1-70k and SF3a60 are reduced by mutation to levels that, in the case of SF3a60, demonstrably interfere with Sxl autoregulation. The observation that increased snf+ does not suppress other phenotypes associated with mutations that reduce U1-70k or SF3a60 is additional evidence that snf+ dose effects are not caused by increased snRNP levels. Mammalian U1A protein, like Snf, has a snRNP-independent function.

Apiconuclear Organization of Microtubules Does Not Specify Protein Delivery from the Trans-Golgi Network to Different Membrane Domains in Polarized Epithelial Cells

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25–50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) ∼40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.

A Novel Synaptobrevin/VAMP Homologous Protein (VAMP5) Is Increased during In Vitro Myogenesis and Present in the Plasma Membrane

cDNA clones encoding a novel protein (VAMP5) homologous to synaptobrevins/VAMPs are detected during database searches. The predicted 102–amino acid VAMP5 harbors a 23-residue hydrophobic region near the carboxyl terminus and exhibits an overall amino acid identity of 33% with synaptobrevin/VAMP1 and 2 and cellubrevin. Northern blot analysis reveals that the mRNA for VAMP5 is preferentially expressed in the skeletal muscle and heart, whereas significantly lower levels are detected in several other tissues but not in the brain. During in vitro differentiation (myogenesis) of C2C12 myoblasts into myotubes, the mRNA level for VAMP5 is increased ∼8- to 10-fold. Immunoblot analysis using antibodies specific for VAMP5 shows that the protein levels are also elevated ∼6-fold during in vitro myogenesis of C2C12 cells. Indirect immunofluorescence microscopy and immunoelectron microscopy reveal that VAMP5 is associated with the plasma membrane as well as intracellular perinuclear and peripheral vesicular structures of myotubes. Epitope-tagged versions of VAMP5 are similarly targeted to the plasma membrane.

Proton long-range migration along protein monolayers and its consequences on membrane coupling

It has been shown with lipid layers and more recently with purple membranes that protons have slow surface-to-bulk transfer. This results in long-range proton lateral conduction along membranes. We report here that such lateral transfer can take place along a pure protein film. It is strongly controlled by the packing. Subtle reorganizations of the protein–protein contact can be biological switches between interfacial and delocalized proton pathways between sources and sinks.

Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Iβ or a membrane-bound cGK II/Iβ chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil

The ectodomain of the Ebola virus Gp2 glycoprotein was solubilized with a trimeric, isoleucine zipper derived from GCN4 (pIIGCN4) in place of the hydrophobic fusion peptide at the N terminus. This chimeric molecule forms a trimeric, highly α-helical, and very thermostable molecule, as determined by chemical crosslinking and circular dichroism. Electron microscopy indicates that Gp2 folds into a rod-like structure like influenza HA2 and HIV-1 gp41, providing further evidence that viral fusion proteins from diverse families such as Orthomyxoviridae (Influenza), Retroviridae (HIV-1), and Filoviridae (Ebola) share common structural features, and suggesting a common membrane fusion mechanism.

Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane

Accumulating evidence suggests that the mitochondrial molecular chaperone heat shock protein 60 (hsp60) also can localize in extramitochondrial sites. However, direct evidence that hsp60 functions as a chaperone outside of mitochondria is presently lacking. A 60-kDa protein that is present in the plasma membrane of a human leukemic CD4+ CEM-SS T cell line and is phosphorylated by protein kinase A (PKA) was identified as hsp60. An 18-kDa plasma membrane-associated protein coimmunoprecipitated with hsp60 and was identified as histone 2B (H2B). Hsp60 physically associated with H2B when both molecules were in their dephospho forms. By contrast, PKA-catalyzed phosphorylation of both hsp60 and H2B caused dissociation of H2B from hsp60 and loss of H2B from the plasma membrane of intact T cells. These results suggest that (i) hsp60 and H2B can localize in the T cell plasma membrane; (ii) hsp60 functions as a molecular chaperone for H2B; and (iii) PKA-catalyzed phosphorylation of both hsp60 and H2B appears to regulate the attachment of H2B to hsp60. We propose a model in which phosphorylation/dephosphorylation regulates chaperoning of H2B by hsp60 in the plasma membrane.

Molecular cloning of Ian4: a BCR/ABL-induced gene that encodes an outer membrane mitochondrial protein with GTP-binding activity

Using the representation difference analysis technique, we have identified a novel gene, Ian4, which is preferentially expressed in hematopoietic precursor 32D cells transfected with wild-type versus mutant forms of the Bcr/Abl oncogene. Ian4 expression was undetectable in 32D cells transfected with v-src, oncogenic Ha-ras or v-Abl. Murine Ian4 maps to chromosome 6, 25 cM from the centromere. The Ian4 mRNA contains two open reading frames (ORFs) separated by 5 nt. The first ORF has the potential to encode for a polypeptide of 67 amino acids without apparent homology to known proteins. The second ORF encodes a protein of 301 amino acids with a GTP/ATP-binding site in the N-terminus and a hydrophobic domain in the extreme C-terminus. The IAN-4 protein resides in the mitochondrial outer membrane and the last 20 amino acids are necessary for this localization. The IAN-4 protein has GTP-binding activity and shares sequence homology with a novel family of putative GTP-binding proteins: the immuno-associated nucleotide (IAN) family.

Depletion of Acyl-Coenzyme A-Binding Protein Affects Sphingolipid Synthesis and Causes Vesicle Accumulation and Membrane Defects in Saccharomyces cerevisiae

Deletion of the yeast gene ACB1 encoding Acb1p, the yeast homologue of the acyl-CoA-binding protein (ACBP), resulted in a slower growing phenotype that adapted into a faster growing phenotype with a frequency >1:105. A conditional knockout strain (Y700pGAL1-ACB1) with the ACB1 gene under control of the GAL1 promoter exhibited an altered acyl-CoA profile with a threefold increase in the relative content of C18:0-CoA, without affecting total acyl-CoA level as previously reported for an adapted acb1Δ strain. Depletion of Acb1p did not affect the general phospholipid pattern, the rate of phospholipid synthesis, or the turnover of individual phospholipid classes, indicating that Acb1p is not required for general glycerolipid synthesis. In contrast, cells depleted for Acb1p showed a dramatically reduced content of C26:0 in total fatty acids and the sphingolipid synthesis was reduced by 50–70%. The reduced incorporation of [3H]myo-inositol into sphingolipids was due to a reduced incorporation into inositol-phosphoceramide and mannose-inositol-phosphoceramide only, a pattern that is characteristic for cells with aberrant endoplasmic reticulum to Golgi transport. The plasma membrane of the Acb1p-depleted strain contained increased levels of inositol-phosphoceramide and mannose-inositol-phosphoceramide and lysophospholipids. Acb1p-depleted cells accumulated 50- to 60-nm vesicles and autophagocytotic like bodies and showed strongly perturbed plasma membrane structures. The present results strongly suggest that Acb1p plays an important role in fatty acid elongation and membrane assembly and organization.

An Intact Dilysine-like Motif in the Carboxyl Terminus of MAL Is Required for Normal Apical Transport of the Influenza Virus Hemagglutinin Cargo Protein in Epithelial Madin-Darby Canine Kidney Cells

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine −3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.

Membrane fusion protein synexin (annexin VII) as a Ca2+/GTP sensor in exocytotic secretion.

Exocytotic membrane fusion and secretion are promoted by the concerted action of GTP and Ca2+, although the precise site(s) of action in the process are not presently known. However, the calcium-dependent membrane fusion reaction driven by synexin (annexin VII) is an in vitro model for this process, which we have now found to be further activated by GTP. The mechanism of fusion activation depends on the unique ability of synexin to bind and hydrolyze GTP in a calcium-dependent manner, both in vitro and in vivo in streptolysin O-permeabilized chromaffin cells. The required [Ca2+] for GTP binding by synexin is in the range of 50-200 microM, which is known to occur at exocytotic sites in chromaffin cells, neurons, and other cell types. Previous immunolocalization studies place synexin at exocytotic sites in chromaffin cells, and we conclude that synexin is an atypical G protein that may be responsible for both detecting and mediating the Ca2+/GTP signal for exocytotic membrane fusion.