Formation Of Membrane Domains Research Articles

Membrane Reorganization during Chilling: Implications for Long-Term Stabilization of Platelets

This essay is a review of the various biophysical and biochemical events that make up the factors responsible for platelet cold-induced activation. It describes the formation of large membrane domains composed of raft aggregates that occur during chilling and storage. It also presents strong evidence that platelet membranes undergo lateral phase separation during prolonged storage in the cold and suggests that raft aggregation and lateral phase separation are key events which must be obviated to stabilize platelets and store them either in the frozen or in the dry state.

Imaging domains in model membranes with atomic force microscopy

Lateral segregation in biomembranes can lead to the formation of biologically functional domains. This paper reviews atomic force microscopy studies on domain formation in model membranes, with special emphasis on transbilayer asymmetry, and on lateral domains induced by lipid–lipid interactions or by peptide–lipid interactions.

Protein-induced formation of cholesterol-rich domains.

A major protein of neuronal rafts, NAP-22, binds specifically to cholesterol. We demonstrate by circular dichroism that NAP-22 contains a significant amount of beta-structure that is not sensitive to binding of the protein to membranes, suggesting that a major portion of the protein does not insert deeply into the membrane. The free energy of binding of NAP-22 to liposomes of dioleoylphosphatidylcholine with 40% cholesterol is -7.3 +/- 0.5 kcal/mol. NAP-22 mixed with dipalmitoylphosphatidylcholine and 40% cholesterol partitions into the detergent insoluble fraction in the presence of 1% Triton X-100. NAP-22 also causes this insoluble fraction to become enriched in cholesterol relative to phospholipid, again demonstrating the ability of this protein to segregate cholesterol and phospholipids into domains. Differential scanning calorimetry results demonstrate that NAP-22 promotes domain formation in liposomes composed of cholesterol and phosphatidylcholine. This is shown by NAP-22-promoted changes in the shape and enthalpy of the phase transition of phosphatidylcholine as well as by the appearance of cholesterol crystallite transitions in membranes composed of phosphatidylcholine with either saturated or unsaturated acyl chains. In situ atomic force microscopy revealed a marked change in the surface morphology of a supported bilayer of dioleoylphosphatidylcholine with 0.4 mole fraction of cholesterol upon addition of NAP-22. Prior to the addition of the protein, the bilayer appears to be a molecularly smooth structure with uniform thickness. Addition of NAP-22 resulted in the rapid formation of localized raised bilayer domains. Remarkably, there was no gross disruption or erosion of the bilayer but rather simply an apparent rearrangement of the lipid bilayer structure due to the interaction of NAP-22 with the lipid. Our results demonstrate that NAP-22 can induce the formation of cholesterol-rich domains in membranes. This is likely to be relevant in neuronal membrane domains that are rich in NAP-22.

Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol.

The ESR spectra from different positional isomers of sphingomyelin and phosphatidylcholine spin-labeled in their acyl chain have been studied in sphingomyelin(cerebroside)-phosphatidylcholine mixed membranes that contain cholesterol. The aim was to investigate mechanisms by which cholesterol could stabilize possible domain formation in sphingolipid-glycerolipid membranes. The outer hyperfine splittings in the ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled on the 5 C atom of the acyl chain were consistent with mixing of the components, but the perturbations on adding cholesterol were greater in the membranes containing sphingomyelin than in those containing phosphatidylcholine. Infrared spectra of the amide I band of egg sphingomyelin were shifted and broadened in the presence of cholesterol to a greater extent than the carbonyl band of phosphatidylcholine, which was affected very little by cholesterol. Two-component ESR spectra were observed from lipids spin-labeled on the 14 C atom of the acyl chain in cholesterol-containing membranes composed of sphingolipids, with or without glycerolipids (sphingomyelin/cerebroside and sphingomyelin/cerebroside/phosphatidylcholine mixtures). These results indicate the existence of gel-phase domains in otherwise liquid-ordered membranes that contain cholesterol. In the gel phase of egg sphingomyelin, the outer hyperfine splittings of sphingomyelin spin-labeled on the 14-C atom of the acyl chain are smaller than those for the corresponding spin-labeled phosphatidylcholine. In the presence of cholesterol, this situation is reversed; the outer splitting of 14-C spin-labeled sphingomyelin is then greater than that of 14-C spin-labeled phosphatidylcholine. This result provides some support for the suggestion that transbilayer interdigitation induced by cholesterol stabilizes the coexistence of gel-phase and "liquid-ordered" domains in membranes containing sphingolipids.

Simulation studies of high-field EPR spectra of spin-labeled lipids in membranes.

The high-field (i.e., 94 GHz) membrane EPR spectra of lipids spin labeled in their fatty acid chains have been simulated by using two limiting motional models. The aim was to identify the dynamic origin of the residual (g(xx) - g(yy)) anisotropy observed in the nonaxial EPR spectra of cholesterol-containing membranes. It is concluded that the residual spectral anisotropy arises from in-plane ordering of the lipid chains by cholesterol. The partial averaging of the (g(xx) - g(yy)) anisotropy was best described by restricted axial rotation with a frequency in the region of tau(-1)(R||) approximately 0.5-1 x 10(9) s(-1). Simulations for slower axial rotation of unrestricted amplitude produced less satisfactory fits. In phospholipid membranes not containing cholesterol, the nonaxial anisotropy is completely averaged in the fluid phase and substantially reduced even in the gel phase. The unrestricted axial rotation in the gel phase is of comparable frequency to that of the limited axial rotation in the liquid-ordered phase of membranes containing cholesterol. These results on in-plane ordering by cholesterol in the liquid-ordered phase could be significant for current proposals regarding domain formation in cellular membranes.

Domain Growth, Shapes, and Topology in Cationic Lipid Bilayers on Mica by Fluorescence and Atomic Force Microscopy

Domain formation in mica-supported cationic bilayers of dipalmitoyltrimethylammoniumpropane (DPTAP) and dimyristoyltrimethylammoniumpropane (DMTAP), fluorescently doped with an NBD (((7-nitro-2–1, 3-benzoxadiazol-4-yl)amino)caproyl) phospholipid, was investigated with fluorescence microscopy and atomic force microscopy. Heating above the acyl chain melting temperature and cooling to room temperature resulted in nucleation and growth of domains with distinguishable patterns. Fractal patterns were found for DPTAP, whereas DMTAP domains were elongated and triangular with feathery edges. Reducing the cooling rate or probe concentration for DPTAP bilayers resulted in larger, filled-in domains with more rounded edges. However, for DMTAP, cooling rates mainly affected size and only slightly modified domain morphology. In a saline environment, the domains were dark, and the surrounding continuous region was bright and thus contained the fluorescent probe. However, as the salt concentration was decreased, the dark regions percolated (connected), resulting in bright domains. Atomic force microscopy scans along domain edges revealed that the dark regions in fluorescence images were approximately 1.4 nm thicker than the light regions. Additionally, the dark regions were of bilayer thickness, approximately 4 nm. Comparison of these results in bilayers to well-documented behavior in Langmuir monolayers has revealed many similarities (and some differences) and is therefore useful for understanding our observations and identifying possible growth mechanisms that may occur in domain formation in cell membranes or supported membrane systems.

Rabenosyn-5, a novel Rab5 effector, is complexed with hVPS45 and recruited to endosomes through a FYVE finger domain.

Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase-dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

Developmental patterns of mST3GaIV mRNA expression in the mouse:In situ hybridization using DIG-labeled RNA probes

mST3GalV synthesizes ganglioside GM3, the precursor for simple and complex a- and b- series gangliosides, and the expression and regulation of mST3GalV (CMP-NeuAc: lactosylceramide alpha2,3-sialyltransferase) activity is central to the production of almost all gangliosides, a class of glycosphingolipids implicated in variety of cellular processes such as transmembrane signaling, synaptic transmission, specialized membrane domain formation and cell-cell interactions. To understand the developmental expression of mST3GalV in mice, we investigated the spatial and temporal expression of mST3GalV mRNA during the mouse embryogenesis [embryonic (E) days; E9, E11, E13, E15] by in situ hybridization with digoxigenin-labeled RNA probes. All tissues from E9 and E11 were positive for mST3GalV mRNA. On E13, mST3GalV mRNA was expressed in various neural and non-neural tissues. In contrast to these, on E15, the telencephalon and liver produced a strong expression of mST3Gal V which was a quite similar to that of E13. In this stage, mST3GalV mRNA was also expressed in some non-neural tissues. These data indicate that mST3GalV is differently expressed at developmental stages of embryo, and this may be importantly related with regulation of organogenesis in mice.

Lipid-binding proteins as stabilizers of membrane microdomains--possible physiological significance.

Below the melting point temperature of lipids, artificial lipid membranes usually exist in the ordered gel phase. Above these temperatures lipid acyl chains become fluid and disordered (liquid-crystalline phase). Depending on the chemical composition of artificial membranes, phase separation may occur, leading to the formation of transient or stable membrane domains. A similar phase separation of lipids into ordered and disordered domains has been observed in natural membranes at physiological temperature range. Moreover, it has been reported that certain proteins prefer certain organization of lipids, as for example glycosylphosphatidylinositol-anchored proteins or Src family of tyrosine kinases. The aim of present review is to discuss the possibility that some lipid microdomains are induced or stabilized by lipid-binding proteins that under certain conditions, for example due to a rise of cytosolic Ca2+ or pH changes, may attach to the membrane surface, inducing clustering of lipid molecules and creation of ordered lipid microdomains. These domains may than attract other cytosolic proteins, either enzymes or regulatory proteins. It is, therefore, postulated that lipid microdomains play important roles within a cell, in signal transduction and enzymatic catalysis, and also in various pathological states, as Alzheimer's disease, anti-phosphatidylserine syndrome, or development of multidrug resistance of cancer cells.

Mixed membranes of sphingolipids and glycerolipids as studied by spin-label ESR spectroscopy. A search for domain formation.

The temperature dependences of the ESR spectra from different positional isomers of sphingomyelin and of phosphatidylcholine spin-labeled in their acyl chain have been compared in mixed membranes composed of sphingolipids and glycerolipids. The purpose of the study was to identify the possible formation of sphingolipid-rich in-plane membrane domains. The principal mixtures that were studied contained sphingomyelin and the corresponding glycerolipid phosphatidylcholine, both from egg yolk. Other sphingolipids that were investigated were brain cerebrosides and brain gangliosides, in addition to sphingomyelins from brain and milk. The outer hyperfine splittings in the ESR spectra of sphingomyelin and of phosphatidylcholine spin-labeled on C-5 of the acyl chain were consistent with mixing of the sphingolipid and glycerolipid components, in fluid-phase membranes. In the gel phase of egg sphingomyelin and its mixtures with phosphatidylcholine, the outer hyperfine splittings of sphingomyelin spin-labeled at C-14 of the acyl chain of sphingomyelin are smaller than those of the corresponding sn-2 chain spin-labeled phosphatidylcholine. This is in contrast to the situation with sphingomyelin and phosphatidylcholine spin-labeled at C-5, for which the outer hyperfine splitting is always greater for the spin-labeled sphingomyelin. The behavior of the C-14 spin-labels is attributed to a different geometry of the acyl chain attachments of the sphingolipids and glycerolipids that is consistent with their respective crystal structures. The two-component ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled at C-14 of the acyl chain directly demonstrate a broad two-phase region with coexisting gel and fluid domains in sphingolipid mixtures with phosphatidylcholine. Domain formation in membranes composed of sphingolipids and glycerolipids alone is related primarily to the higher chain-melting transition temperature of the sphingolipid component.

Polyelectrolyte molecular weight and electrostatically-induced domains in lipid bilayer membranes.

Polyelectrolyte-induced domain formation in charged lipid bilayer membranes was investigated as a function of polyelectrolyte molecular weight using 2H nuclear magnetic resonance (NMR) spectroscopy. Lipid bilayers consisting of mixtures of alpha- or beta-choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC-alpha-d2 or POPC-beta-d2) plus the cationic amphiphile 1,2-dioleoyl-3-(dimethylamino)propane (DODAP) were exposed to the anionic polyelectrolyte poly(sodium 4-styrenesulfonate) (PSSS) of various molecular weights. Regardless of molecular weight, PSSS produced dual component 2H NMR spectra, indicating two distinct POPC populations, corresponding to PSSS-bound and PSSS-free lipid, in slow exchange with one another. Analysis of the 2H NMR subspectra quadrupolar splittings and intensities showed the PSSS-bound domain to be enriched in DODAP, with the PSSS-free domain correspondingly depleted. At polyelectrolyte loadings below global charge equivalence, PSSS bound DODAP stoichiometrically for all PSSS molecular weights, indicating that the polyelectrolyte chain lies flat upon the membrane surface. At higher PSSS loadings the domains dissipated, leading to single component 2H NMR spectra. At high NaCl concentrations PSSS dissociated from the bilayer surface. Domain size on a per PSSS chain basis increased while the degree of enrichment with DODAP decreased progressively as the PSSS chain length decreased. Such molecular weight-dependent domain characteristics have not been predicted theoretically and need to be taken into account in future refinements of domain models.

2H NMR and polyelectrolyte-induced domains in lipid bilayers

2H NMR studies of polyelectrolyte-induced domain formation in lipid bilayer membranes are reviewed. The 2H NMR spectrum of choline-deuterated phosphatidylcholine (PC) reports on any and all sources of lipid bilayer surface charge, since these produce a conformation change in the choline head group of PC, manifest as a change in the 2H NMR quadrupolar splitting. In addition, homogeneous and inhomogeneous surface charge distributions are differentiated. Adding polyelectrolytes to lipid bilayers consisting of mixtures of oppositely charged and zwitterionic lipids produces 2H NMR spectra which are superpositions of two Pake sub-spectra: one corresponding to a polyelectrolyte-bound lipid population and the other to a polyelectrolyte-free lipid population. Quantitative analysis of the quadrupolar splittings and spectral intensities of the two sub-spectra indicate that the polyelectrolyte-bound populations is enriched with oppositely charged lipid, while the polyelectrolyte-free lipid population is correspondingly depleted. The same domain-segregation effect is produced whether cationic polyelectrolytes are added to anionic lipid bilayers or anionic polyelectrolytes are added to cationic lipid bilayers. The 2H NMR spectra permit a complete characterization of domain composition and size. The anion:cation ratio within the domains is always stoichiometric, as expected for a process driven by Coulombic interactions. The zwitterionic lipid content of the domains is always statistical, reflecting the systems tendency to minimize the entropic cost of demixing charged lipids into domains. Domain formation is observed even with rather short polyelectrolytes, suggesting that individual polyelectrolyte chains aggregate at the surface to form “superdomains”. Overall, the polyelectrolyte bound at the lipid bilayer surface appears to lie flat along the surface and to be essentially immobilized through its multiple electrostatic contacts.

Thermodynamics of Lipid Organization and Domain Formation in Phospholipid Bilayers

Lipid organization and domain formation in phospholipid membranes has a large impact on a number of physical membrane properties such as membrane elasticity, lipid lateral diffusion, permeability, and binding properties for peptides and proteins. The mixing behavior of lipids can be deduced from the binary phase diagram, i.e. temperature versus mole fraction diagram, describing the temperature induced phase transition behavior of lipid mixtures. An easy way to obtain experimental data to construct a binary phase diagram is differential scanning calorimetry (DSC) of aqueous dispersions of liposomes of lipid mixtures. The temperatures that correspond to points on the liquidus and solidus curves of the phase diagram are determined from the DSC thermograms, e.g. the experimental heat capacity curves. The main problem to solve is the correct determination of the onset and end of the melting curve for every single transition curve in a standardized manner, because of the lack of theoretically justified methods. To avoid this subjective procedure, we have developed a method for the simulation of heat capacity curves that are obtained by DSC. Our model uses a thermodynamic approach for the description of the miscibility of phospholipids, which is based on regular solution theory with an incorporation of a broadening function, taking into account the reduced cooperativity in mixed lipid bilayer systems. The output parameters of these simulations are nonideality parameters describing nonideal mixing in the gel and liquid crystalline phase, the temperature set describing the onset and end of melting, respectively, and a value for the cooperative unit size. These temperature data are then used for the construction and simulation of the phase diagrams. A four parameter model was used, taking into account the asymmetry of the mixing behavior, yielding two nonideality parameters for each phase. These nonideality parameters describe the interaction of both components in the single phase regions. We have employed this procedure to describe the mixing behavior of various binary phospholipid systems. We will show how the mixing behavior is influenced by changing the hydrocarbon chain length of one of the component, by changing the lipid-lipid head group interaction, or the pH and ionic strength of the dispersion. Examples of binary lipid mixtures in which domain formation in the liquid crystalline phase is observed, are presented.

Division-associated changes in membrane viscosity of Escherichia coli

Septum formation is initiated by the FtsZ ring assembly in the middle of rod-shape bacteria. The mechanism which determines the division site in the membrane and makes it recognizable by FtsZ is still unknown. We have recently demonstrated that the putative division membrane domains can be visualized by a fluorescent membrane probe (Fishov and Woldring, Mol. Microbiol., 1999) and that these domains can be dissipated by interrupting the process of coupled transcription and translation of proteins (Binenbaum et al., Mol. Microbiol., 1999). Here, we examined the membrane dynamics of Escherichia coli during division and after a reversible division arrest. Anisotropy of DPH fluorescence, used as an indicator of membrane dynamics (viscosity), correlated with the rate of division in synchronous cells. It decreased during filamentation caused by drugs or by temperature, but not in the ftsZ mutant and when DNA replication was blocked by nalidixic acid. Based on previous data, we incline to interpret these results as reflecting formation and dissipation of putative membrane domains marking the division sites; domains are formed by partitioning nucleoids and dissipate while used for constriction or after the nucleoids have been segregated too far in a filament.

Division-associated changes in membrane viscosity of Escherichia coli

Septum formation is initiated by the FtsZ ring assembly in the middle of rod-shape bacteria. The mechanism which determines the division site in the membrane and makes it recognizable by FtsZ is still unknown. We have recently demonstrated that the putative division membrane domains can be visualized by a fluorescent membrane probe (Fishov and Woldring, Mol. Microbiol., 1999) and that these domains can be dissipated by interrupting the process of coupled transcription and translation of proteins (Binenbaum et al., Mol. Microbiol., 1999). Here, we examined the membrane dynamics of Escherichia coli during division and after a reversible division arrest. Anisotropy of DPH fluorescence, used as an indicator of membrane dynamics (viscosity), correlated with the rate of division in synchronous cells. It decreased during filamentation caused by drugs or by temperature, but not in the ftsZ mutant and when DNA replication was blocked by nalidixic acid. Based on previous data, we incline to interpret these results as reflecting formation and dissipation of putative membrane domains marking the division sites; domains are formed by partitioning nucleoids and dissipate while used for constriction or after the nucleoids have been segregated too far in a filament.

Transcription- and translation-dependent changes in membrane dynamics in bacteria: testing the transertion model for domain formation.

Cell cycle events have been proposed to be triggered by the formation of membrane domains in the process of coupled transcription, translation and insertion ('transertion') of nascent membrane and exported proteins. Disruption of domain structure should lead to changes in membrane dynamics. Membrane viscosity of Escherichia coli and Bacillus subtilis decreased after inhibition of protein synthesis by chloramphenicol or puromycin, or of RNA initiation by rifampicin, but not after inhibition of RNA elongation by streptolydigin or amino acid starvation of a stringent strain. The decrease caused by inhibitors of protein synthesis was prevented by streptolydigin if added simultaneously, but was not reversed if added later. The drug-induced decrease in membrane viscosity is energy dependent: it did not happen in KCN-treated cells. All treatments decreasing membrane viscosity also induced nucleoid compaction and fusion. Inhibition of macromolecular synthesis without membrane perturbation caused nucleoids to expand. Changes in membrane dynamics were also displayed during a nutritional shift-down transition that causes imbalance in macromolecular syntheses. The results are consistent with the transertion model, predicting dissipation of membrane domains by termination of protein synthesis or detachment of polysomes from DNA; domain structure is conserved if the transertion process is 'frozen'.

Elf3 encodes a novel 200-kD beta-spectrin: role in liver development.

beta-spectrins are crucial for the maintenance of cell shape, the establishment of cell polarity, and the formation of distinct membrane domains. Our strategy for identifying genes important for hepatocyte polarity has been to utilize subtractive hybridization of early embryonic mouse cDNA liver libraries. As a result, we have cloned three isoforms of a novel beta-spectrin elf (embryonic liver beta-fodrin), and here we report the analysis of elf3, the longest isoform (8172 nt). ELF3 comprises 2154 residues with an overall similarity of 89.0% and 95.3% to mouse beta-spectrin (betaSpIIsigma1) at the nucleotide and amino acid level, respectively. ELF3 is characterized by an actin-binding domain, a long repeat domain, and a short regulatory domain remarkable for the absence of a PH domain. Linkage analysis reveals that elf3 maps to mouse chromosome 11 between D11Bir6 and D11Xrf477, a different chromosomal locus from that of the other four spectrin genes. Northern blot analysis utilizing an elf3 3'-UTR probe demonstrates an abundant 9.0-kb transcript in brain, liver, and heart tissues. Western blot with a polyclonal antibody against ELF identifies a 200 kD protein in mouse liver, brain, kidney, and heart tissues. Immunohistochemical studies demonstrate ELF labeling of the basolateral or sinusoidal membranes surface as well as a granular cytoplasmic pattern in hepatocytes. Antisense studies utilizing cultured liver explants show a vital role of elf3 in hepatocyte differentiation and intrahepatic bile duct formation. The differential expression, tissue localization, and functional studies demonstrate the importance of elf3 in modulating interactions between various components of the cytoskeleton proteins controlling liver and bile duct development.

Membrane domain formation by calcium-dependent, lipid-binding proteins: insights from the C2 motif

We propose a novel role in cellular function for some membrane-binding proteins and, specifically, the C2 motif. The C2 motif binds phospholipid in a manner that is modulated by Ca 2+ and is thought to confer membrane-binding ability on a wide variety of proteins, primarily proteins involved in signal transduction and membrane trafficking events. We hypothesize that in the absence of Ca 2+ the C2 motif couples the free energy of binding to a bilayer membrane comprised of zwitterionic and negatively charged lipids to the formation of a domain enriched in the negative lipids. This in turn leads to the dynamic clustering of bound homologous or heterologous proteins incorporating the C2 motif, or other acidic lipid-binding motifs. In the presence of Ca 2+, the protein clusters may be further stabilized. In support of this hypothesis we present evidence for membrane domain formation by the first C2 domain of synaptotagmin in the absence of Ca 2+. Fluid state phospholipid mixtures incorporating a pyrene-labeled phospholipid probe exhibited a change in pyrene excimer/monomer fluorescence ratio consistent with domain formation upon binding the C2 domain. In addition, we present the results of simulations of the interaction of the C2 domain with the membrane that indicate that protein clusters and lipid domains form in concert.

A spectrin membrane skeleton of the Golgi complex

The existence of a Golgi-localized membrane cytoskeleton has been revealed by the identification of two major components of the spectrin membrane skeleton, spectrin and ankyrin, that associate with the Golgi complex. Golgi spectrin was identified with an antibody specific for the β-subunit of the erythroid isoform of spectrin (β1Σ1). This antibody recognizes a 220 kDa polypeptide that localizes to discrete regions of the Golgi complex and associates with Golgi membranes in a Brefeldin A sensitive manner. Two isoforms of Golgi ankyrin have been identified: a 119 kDa form (Ank G119) which represents a truncated, alternatively spliced isoform of a recently cloned novel ankyrin of the nervous system Ank G, and a larger 195 kDa ankyrin (Ank 195) that cross-reacts with antibodies to erythrocyte ankyrin. A Golgi localized membrane skeleton composed of these unique membrane skeleton isoforms could serve a variety of important functions, including the maintenance of Golgi structural organization and the formation of discrete membrane domains within Golgi compartments.

The role of ankyrin and spectrin in membrane transport and domain formation

Recent discoveries reveal a Golgi-centric spectrin—ankyrin skeleton required for Golgi integrity and anterograde protein trafficking. Identification of specific functional domains in spectrin that mediate its association with motor proteins and the Golgi complex has allowed novel insights into the structure and function of the secretory pathway, and into how this process is controlled by ADP-ribosylation factor and phosphoinositides. Alternative models of Golgi spectrin function that have been recently proposed are reviewed.