INTRODUCTION

A fundamental and striking property of eukaryotic cells is theirability to dynamically organize and maintain morphologicallydistinct membrane-bounded compartments while selectivelytransporting newly synthesized or recycled lipids and proteinsthrough secretory and endocytic pathways. Recent discoveriesattribute an increasingly important role to cytoskeletal proteinsin this process. Cytoskeletal proteins serve two fundamentalfunctions in membrane trafficking: organelle motility (inconjunction with motor proteins), and membrane-organization(in conjunction with adapter and integral membrane proteins).The outlines of how some cytoskeletal elements contribute tothe motility of organelles or transport intermediates along theendocytic and exocytic pathways are well described – forexample, the way that the microtubule system and itsassociated motors mediate long-distance movement (Holleranand Holzbaur, 1998; Lippincott-Schwartz, 1998), or the waythat the actin system and actin-based motors effect shorter-range movement (Stow et al., 1998, Allan and Schroer, 1999;Fath et al., 1994; Heimann et al., 1999). However, much lessis known about the way that organelles or transport containers

are themselves organized, stabilized, shaped, or linked to themotors of transport.

Spectrin is a cytoskeletal protein that can control membraneorganization, stability and shape, and link membranes to themotors of transport as well as to all major filament systems.Spectrin has also recently emerged as a participant in thesecretory pathway (for earlier reviews, see Beck and Nelson,1998; De Matteis and Morrow, 1998). Various isoforms ofspectrin and its key adapter protein ankyrin are present on theGolgi, on transport intermediates, and on the membranes of theendo-lysosomal pathway. Disruption of spectrin function eitherby dominant negative spectrin mutants or by anti-spectrinantibodies blocks anterograde transport in the secretory pathway,and direct regulation of the interaction between spectrin andGolgi membranes by the small G protein ADP-ribosylationfactor (ARF) is well established. Although much of the work onspectrin in the secretory and endocytic pathways is recent, andsome has only appeared in abstract form, consideration of thesefindings in the light of spectrin’s well-studied role at the plasmamembrane yields a comprehensible picture of the uniqueproperties that spectrin brings to the Golgi and other organelles.These considerations are the focus of this review.

2331Journal of Cell Science 113, 2331-2343 (2000)Printed in Great Britain © The Company of Biologists Limited 2000JCS0780

The paradox of how the Golgi and other organelles can sorta continuous flux of protein and lipid but maintaintemporal and morphological stability remains unresolved.Recent discoveries highlight a role for the cytoskeleton inguiding the structure and dynamics of organelles. Perhapsone of the more striking, albeit less expected, of thesediscoveries is the recognition that a spectrin skeletonassociates with many organelles and contributes to themaintenance of Golgi structure and the efficiency of proteintrafficking in the early secretory pathway. Spectrininteracts directly with phosphoinositides and withmembrane proteins. The small GTPase ARF, a key playerin Golgi dynamics, regulates the assembly of the Golgispectrin skeleton through its ability to controlphosphoinositide levels in Golgi membranes, whereasadapter molecules such as ankyrin link spectrin to other

membrane proteins. Direct interactions of spectrin withactin and centractin (ARP1) provide a link to dynein,myosin and presumably other motors involved withintracellular transport. Building on the recognized abilityof spectrin to organize macromolecular complexes ofmembrane and cytosolic proteins into a multifacetedscaffold linked to filamentous structural elements (termedlinked mosaics), recent evidence supports a similar role forspectrin in organelle function and the secretory pathway.Two working models accommodate much of the availabledata: the Golgi mesh hypothesis and the spectrin ankyrinadapter protein tethering system (SAATS) hypothesis.

Key words: Cytoskeleton, Cargo selection, Linked mosaic, Micro-domain, Actin, Protein sorting, Ankyrin, ARF, SAATS, Golgiscaffold

SUMMARY

COMMENTARY

Spectrin tethers and mesh in the biosynthetic pathway

Maria Antonietta De Matteis 1,* and Jon S. Morrow 2,*1Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro (Chieti), Italy 2Department of Pathology and the Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven,Connecticut, USA*Correspondence may be addressed to either author (e-mail: demattei@cmns.mnegri.it; jon.morrow@yale.edu)

Published on WWW 14 June 2000

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THE SPECTRIN MEMBRANE SKELETON:EXTENSIBLE LINKED MOSAICS

First identified as the supporting infrastructure of the plasmamembrane of erythrocytes, spectrin is now recognized as themost central player in a ubiquitous and complex linkagebetween membranes and the cytosol. By bindingsimultaneously to integral membrane proteins, cytosolicproteins and certain phospholipids, either directly or throughadapter proteins, spectrin creates a multifunctional scaffold atthe membrane interface on which macromolecular complexesof membrane proteins, cytoplasmic signaling molecules, andstructural elements are organized (Fig. 1). In addition, spectrinbinds (either directly or via adapter or motor proteins) to allmajor filament systems and thereby links mosaics or islandsof membrane and cytosolic proteins to cytoskeletal elements.These properties have led to the general concept of the spectrinmembrane skeleton as a series of ‘linked mosaics’ (De Matteisand Morrow, 1998; Morrow et al., 1997). The characteristicsof the skeleton may vary: in the archetypal erythrocyteskeleton, the mosaics are frequent, homogeneous, and joinedby short actin filaments so as to form a homogeneous quasi-hexagonal lattice. Alternatively, the mosaics can be sparse andlinked to longer microfilaments or to microtubules viadynein/dynactin, forming the types of spectrin skeletonassociated with organized receptor clusters or membranemicrodomains. Examples of such structures include thespectrin skeleton found at the neuronal post-synaptic density(Malchiodi-Albedi et al., 1993; Wechsler and Teichberg,1998), at the nodes of Ranvier (S. Berghs et al., unpublished;Lambert et al., 1997), at the acetylcholine receptor cluster ofskeletal muscle (Pumplin, 1995), at the basolateral infoldingsof epithelial cells (Drenckhahn and Merte, 1987), and perhapsthe spectrin complex found on tubular-vesicular transportintermediates, the Golgi and other organelles (see below).

But there is more to the spectrin skeleton. A separate conceptthat evolved from studies of the erythrocyte identifies spectrinas a type of molecular spring or shock-absorber, a moleculethat not only tethers integral and cytosolic proteins but alsocontrols their spacing by altering its flexibility and contourlength. Previously, this notion found support only inbiophysical studies that detected altered elasticity of themembrane as a function of spectrin perturbation (Stokke et al.,1986), in the significant differences in scaled flexural rigidity(a measure of stiffness) between different types of spectrin(Coleman et al., 1989), and in morphological studies thatrevealed the in situ length of spectrin to be variable andmuch shorter than the 2000-Å length of extended spectrinheterodimers (McGough and Josephs, 1990; Ursitti et al.,1991). Grum et al. (1999) have now elegantly revealed thestructural basis of this unusual property. A defining feature ofspectrin and spectrin-related proteins (including α-actinin anddystrophin) is the presence of many tandem, antiparallelcoiled-coil repeats. Whereas the features of a single repeat arepredictable on the basis of its α-helical content and have beenconfirmed (Yan et al., 1993), it is the linker region betweenrepeats and the ability of two spectrin chains to supercoil thatappears to control the length and flexibility of spectrin (Fig.1B).

Each spectrin repeat is composed of three helices (A, B andC). Conformational rearrangements involving the helix B-C

loop, variations in the length of the B helix and variations inthe degree of super coiling appear to control both the flexibilityof the link and the overall length of the molecule (Grum et al.,1999). Thus, a more complete concept of the spectrin skeletonis as a series of extensible linked mosaics(Fig. 1C). Thisstructure can accommodate limited membrane deformation,limited diffusional motion and the variable spacing required tocapture a variety of ligands, while adjusting to variations inmembrane curvature and providing larger-scale order andsupport.

Finally, at least at the plasma membrane, all interactionsinvolving spectrin and its adapter molecules, including theelasticity of spectrin itself, appear to be regulated post-translationally. Identified mechanisms include phosphorylationby a variety of kinases, the action of calcium and calmodulin,calcium-activated proteolysis, and regulation by small GTP-binding proteins, pH and myristolyation. In addition, multipleisoforms of spectrin exist (Tables 1 and 2), as do variants thatarise by alternative mRNA splicing that specifically alter thehelix B-C loop and possibly thereby the flexibility or spacingof a sub-region of the molecule (Cianci et al., 1999b).

HOW DOES THE SPECTRIN SKELETON CONTROLMEMBRANE STABILITY AND SHAPE?

Before considering the role of spectrin at the Golgi and in thesecretory pathway, it is worthwhile to ponder its role in the redcell. Specifically, why is spectrin needed to stabilize themembrane, and why in its absence do red cell membranesspontaneously endovesiculate? Early concepts attributed a

M. A. De Matteis and J. S. Morrow

Fig. 1. Spectrin is a multifunctional extended molecule that forms amembrane skeleton composed of extensible linked mosaics.(A) Spectrin usually exists as anti-parallel heterodimers of an α-spectrin and a β-spectrin. Homopolymeric β-spectrin complexesexist in skeletal muscle (Bloch and Morrow, 1989) and possibly theGolgi (Beck et al., 1994; Devarajan et al., 1996). Each subunitdisplays a tripartite organization; non-homologous ends (regions 1and 3) flank a central domain (region 2) composed of multiple ≈106-residue coiled-coil α-helical repeats. Functional specializationsappear within these repeats as indicated. Ligands reported to bindone or more forms of spectrin, and the best-recognized adapterproteins, are shown along with their approximate binding site.(B) The ‘extensible linked mosaic’ model of spectrin action. Thefundamental role of the spectrin skeleton is to control lateral order inthe plane of the membrane (see Fig. 2). Through its capacity to bindmultiple ligands selectively and to self-associate through hetero- andhomo-typic interactions, end on and side to side, spectrin can formordered arrays or mosaics of limited size. Associated with thesenascent arrays are embedded and soluble proteins. The ability ofspectrin to expand or contract, and to alter its flexibility, controls thedensity of packing within each mosaic and possibly local membranecurvature. Mosaics are joined by linking interactions involving F-actin, microtubules or intermediate filaments to form larger arrays.Active redistribution or macro-organization of such mosaics alongmicrofilaments or microtubules is achieved by linkage of the mosaicsto the appropriate motors. (C) The basic structural motif of spectrinis a series of tandem, antiparallel coiled-coil repeats. Two repeats arejoined by a continuation of helix C into helix A of the adjacentrepeat. Conformational rearrangements involving the helix B-C loop,and variations in super-coiling between the two spectrin chains, canrapidly vary the length and flexibility of the molecule (adapted withpermission from Grum et al., 1999).

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mechanical role to spectrin, arguing that it provided theincrement of elasticity and rigidity needed to withstand theturbulence of circulation. A better model focuses on howintegral proteins themselves affect membrane stability andshape. For example, in hereditary ovalocytosis, a nine-residuedeletion in the red cell anion-exchange protein AE1 does notalter the structure or composition of the spectrin skeleton, butyields rigid, ovalocytic red cells (Liu et al., 1995; Mohandaset al., 1992). Similarly, deletion of the AE1gene yields fragilered cells but does not change the spectrin skeleton or itsassociation with the bilayer (Peters et al., 1996; Southgate etal., 1996). Experimentally, the elasticity of the membranedepends as much on integral membrane protein attachments ason any property of the skeletal lattice itself (Sleep et al., 1999).These seemingly disparate observations can be reconciled if

the primary role of spectrin is not to lend rigidity to themembrane directly, but rather to control the lateral distributionof integral membrane proteins. In this view, alterations inmembrane shape and stability arise from processes that affectthe distribution of integral membrane proteins. Examples ofsuch processes include the weakening of contacts between theskeleton and membrane proteins (as in many forms ofhereditary spherocytosis), the weakening of lateral associationswithin the skeleton (as in hereditary elliptocytosis), oralterations in the abundance or properties of the integralmembrane proteins themselves (as in ovalocytosis).

A more rigorous way to view the role of a peripheralmembrane skeleton and its relationship to integral membraneprotein distributions is to consider a biological membrane as atrilayer consisting of a skeletal layer (the spectrin skeleton, or

M. A. De Matteis and J. S. Morrow

Table 1. Summary of spectrin isoformsSpectrin Human chromosome Pre-Golgi Golgi TGN/endosome/lysosome Plasma membrane Comments

αΙ 1 No No Yes Yes Predominant in red cells; also endo/lysosomal compartment

αΙΙ 9 No No No Yes Generalized plasma membrane α-spectrinβΙ 14 Yes Yes Yes Yes Predominant in red cells (βΙΣ1), also in

brain and muscle (βΙΣ2); immunoreactive with Golgi forms

βΙΙ 2 No No No Yes Generalized plasma membrane β-spectrinβΙΙΙ 11 Yes Yes Yes No (trace) Golgi and transport container associated

spectrin; binds ARP1 and munc13βΙV 19 No peri-nuclear Yes Yes MAD2 domain contains novel ICA/512

secretory granule protein binding domain;associates also with nodes of Ranvier

βV 15 ND ND ND Yes Preserved actin binding and MAD1 domains(Stabach and Morrow, 2000); associates with internal membranes; orthologue of Drosophila beta-heavy

All spectrins have a tripartite structure composed of non-homologous regions 1 and 3 at their termini and a central region 2 composed of a variable number of≈106-residue repeat units (reviewed by Morrow, 1998). Two genes encode α-spectrins that characteristically display an N-terminal β-spectrin-binding site, acentral SH3 domain, and two C-terminal calcium-binding EF-hand motifs. Five genes exist that encode β-spectrins.

Table 2. Summary of ankyrin isoformsAnkyrin Human chromosome Pre-Golgi Golgi TGN/endosome/lysosome Plasma membrane Comments

Ank R 8210 No No No Yes Predominant red cell form. 24 repeat units.

May be a basolateral plasma membrane-associated ankyrin in MDCK cells

SR Yes ?? No No Unusually small transcript of 3′ exon found in skeletal muscle sarcoplasmic reticulum

195 ?? ?? Yes Yes No Not characterized; immunologically similar to AnkR

AnkB 4 Required for intracellular vesicular sorting440 No No Yes Yes 24 repeats; large non-homologous insertion220 No No Yes Yes 24 repeats

AnkG 10 Many isoforms arise by alternative mRNA splicing. Most widely expressed ankyrin family

480 No No No Yes 24 repeats; large non-homologous insertion270 No No Yes Yes 24 repeats; non-homologous insertion190 No No Yes Yes 24 repeats119 Yes Yes Yes No 13 repeats; truncated regulatory domain120 No No Yes No 4 repeats 100 No No Yes No 0 repeats; preserved spectrin-

binding/regulatory domains only

Three ankyrin genes are known, and the encoded proteins display three domains: (i) a highly-conserved N-terminal domain of tandemly arrayed repeats (33-residues each) that bind many proteins; (ii) a well-conserved central domain that binds spectrin; and (iii) a variably sized C-terminal ‘regulatory’ domain that canmodulate the activities of the first two domains. Several novel ankyrin isoforms, some of which lack all or part of the repetitive or regulatory domain, or havelarge inserts, have been described.

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any coat complex, such as coat protein (COP) I or II or clathrin)in close contact with a phospholipid bilayer with its embeddedproteins (Kralj-Iglic et al., 1996; Mohandas and Evans, 1994;Svetina et al., 1996; reviewed by Morrow et al., 1997). The twolipid layers are joined by hydrophobic forces; the skeleton isattached, directly or via adapter proteins, through interactionswith integral membrane proteins or lipids. The energycontributed by the embedded proteins is proportional to theirabundance, the curvature of the membrane, and the strength oftheir interactions with the surrounding bilayer and otherproteins in the bilayer (Kralj-Iglic et al., 1996). At equilibrium,the energy of this system will seek a minimum, and manyintegral membrane proteins will seek to aggregate or beexcluded from regions of increased membrane curvature. Theresult of this tendency is that membrane fragmentation andvesiculation become energetically preferred as theconcentration of protein in the membrane increases (Fig. 2).This tendency is resisted by the favorable energetics ofattaching integral proteins to a homogeneous skeleton.Conversely, discontinuities in the skeleton may favor localinhomogeneities, creating specialized microdomains that canpromote curvature, shape variation, and even vesiculation.Thus, although not all integral membrane proteins will requireactive management by a peripheral skeleton (as born out by therapid diffusional mobility of some resident Golgi proteins, seeCole et al., 1996), one can anticipate that many will – perhapsthose that are more complex, multimeric or bulky.

A necessary feature of above model is that spectrin mustbind, either directly or indirectly, to many integral membraneproteins (see Fig. 1). How can so many different ligands bebound with specificity? Beyond the fact that spectrin is a largeand extended molecule, one answer appears to lie in theremarkable properties of ankyrin, spectrin’s best-understoodadapter. With few exceptions (Table 2), ankyrins typicallypossess a well-conserved spectrin-binding domain coupled to avariable number of 33-residue homologous repeat units. Theankyrin repeat is a generalized protein-protein interaction motiffound in many proteins (for review, see Michaely and Bennett,1992). Each ankyrin repeat forms an L-shaped structureconsisting of a β-hairpin and two α-helices; multiple repeatsassemble to form an oblong structure in which their α-helicalcores are shielded and their β-hairpins are exposed (e.g. seeFoord et al., 1999; Gorina and Pavletich, 1996; Venkataramaniet al., 1998; Yang et al., 1998; Fig. 3). Sequence alignment ofthe repeat units also reveals that the exposed loops varymarkedly, whereas the helices are conserved. The analogy thatcomes to mind is that of a Velcro® ball, a multitude of potentialprotein-binding sites being created by the bristly surface of thehighly variable hairpin loops. For example, AnkG119, the formof ankyrin associated with the Golgi and pre-Golgiintermediates, has 13 ankyrin-repeat units (Devarajan et al.,1996). If one assumes that each of the β-hairpin loops canprovide only a single interaction site (a very conservativeassumption), and that an effective interaction with a proteinligand is generated by any unique combination of three hairpinloops, then AnkG119 could offer unique binding sites to >1700different proteins (one at a time, of course). Although the truevalency of ankyrin is unlikely to be so high, the potential of thissingle family of adapter molecules to bind so many diverseligands is an important consideration as one seeks to understandthe role of these proteins in membrane biogenesis and secretion.

THE COMPOSITION AND ASSEMBLY OF THEGOLGI-ASSOCIATED SPECTRIN SKELETON

SpectrinAlthough many studies have detected spectrin or othercomponents of the spectrin skeleton in association withorganelles (e.g. see Black et al., 1988; De Cesaris et al., 1989;Malchiodi-Albedi et al., 1993; Riederer et al., 1986; Zagon etal., 1986), the exact nature of these associations has onlyrecently been revealed (Beck et al., 1994, 1997; Beck andNelson, 1996, 1998; De Matteis and Morrow, 1998; Devarajanand Morrow, 1996; Devarajan et al., 1996; Godi et al., 1998;Hoock et al., 1997; Stankewich et al., 1998; Ziemnicka-Kotulaet al., 1998). Most antibodies to erythrocyte βΙ spectrinvariably mark by indirect immunofluorescence a perinuclearreticular complex coincident with resident Golgi markers(Beck et al., 1994; Devarajan and Morrow, 1996; Godi et al.,1998). Such antibodies also often stain punctate 500-nm-and-smaller cytoplasmic structures that partially coincide withmarkers of both pre-Golgi as well as post-Golgi compartments(see below; P. Devarajan and J. S. Morrow, and Y. Ch’ng,unpublished; Devarajan and Morrow, 1996; Stankewich et al.,1998). By immunoprecipitation or western blotting, antibodiesto βΙ spectrin detect a 220–240-kDa protein in most culturedcell lines. Immunoelectron microscopy confirms the presenceof spectrin on the Golgi (Beck et al., 1994), althoughrefinement of its ultrastructural characterization has provensurprisingly difficult, and the question of whether specifictypes of spectrin are confined to any specific subcompartmentof the Golgi remains open. Disruption of the Golgi by brefeldinA (BFA) disrupts the Golgi-associated spectrin (Beck et al.,1994; Godi et al., 1998), whereas spectrin remains with theresidual Golgi elements following nocodazole dispersal (Becket al., 1994). Taken together, these results indicate that a βΙ-spectrin-related protein is dynamically associated with theGolgi complex in many (if not all) cells.

Sequences derived from βΙ spectrin can also associate withthe Golgi and function in (or block) the secretory pathway.Full-length βΙΣ1 spectrin (Beck et al., 1994) and recombinantpeptides representing specific βΙΣ1 and βΙΣ2 spectrinsequences bind to the Golgi in cultured MDCK cells(Devarajan et al., 1997a). These studies identify a specificconstitutive Golgi-targeting signal near the N-terminus of βΙspectrin; this sequence includes the actin-binding domain andmembrane-association domain 1 (MAD1; Davis and Bennett,1994; Lombardo et al., 1994). Similar constructs derived fromβΙΙ spectrin sort not to the Golgi but to the plasma membrane(Stabach et al., 1993; Stabach et al., unpublished). A secondpowerful signal that directs the association of spectrin with theGolgi is membrane-association domain 2 (MAD2), whichincludes a pleckstrin homology (PH) domain. Although somespectrins lack this domain (e.g. βΙΣ1 from erythrocytes), mostpossess it (summarized in Table 1).

The MAD2 sequence varies greatly between differentspectrins in this domain (Berghs et al., 1999; Stankewich et al.,1998; Stabach and Morrow, 2000), which presumably reflectsfunctional specialization. At least six MAD2 ligands have beenidentified for different spectrins: βγ G protein signalingsubunits (Wang et al., 1994); D3BP, a very large protein(>400 kDa) of unknown function (Cianci et al., 1997; Li et al.,1998); phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2;

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Hyvonen et al., 1995); the GLUT4 receptor (Corcoran et al.,1997); fodaxin (A60), an axon-specific protein (Hayes et al.,1997); and possibly (although not yet proven) islet cellautoantigen (ICA) 512/IA-2, a receptor tyrosine phosphataseassociated with secretory granules in neuroendocrine cells(Berghs et al., 1999, and unpublished). The first three of theseligands bind to the spectrin PH domain; the others do not.Additional ligands no doubt associate with this domain, andthis is also the site targeted by several kinases (Fig. 1).Although transfection of peptides that have the MAD2 of βΙΣ2spectrin reveals that this domain alone does not concentrate inthe Golgi (Devarajan et al., 1997a), in vitro studies reveal thatit modulates the association of spectrin with isolated Golgifractions (Godi et al., 1998) and that binding is regulated bythe small GTPase ARF, a crucial player in Golgi dynamics.

The role of ARF in the regulation of spectrin dynamics inthe secretory pathway is particularly interesting.As noted above, BFA, a fungal toxin that preventsthe activation of ARF, induces the rapid releaseof spectrin from Golgi membranes (Godi et al.,1998), an action directly related to the ability ofARF to enhance the synthesis of PtdIns(4,5)P2on these membranes (Godi et al., 1998, 1999).The effect of ARF on PtdIns(4,5)P2 synthesisis independent of its ability to stimulatephospholipase D (PLD) or the assembly of COPI.In fact ARF is able to recruit and maintaina specific PI4K isoform, PI4Kβ, and anunidentified PtdInsP5 kinase on Golgimembranes (Godi et al., 1999). The loss of PI4Kβactivity, obtained by transfecting the dominantnegative mutant D656A-PI4Kβ (which is devoidof kinase activity), induces a tubulovesiculartransformation of the Golgi complex.Phosphoinositides such as PtdIns(4,5)P2 thusappear to play both a direct structural role inGolgi membranes, and act as binding sites for

M. A. De Matteis and J. S. Morrow

Fig. 3. The repeat structure of ankyrin forms apolyvalent generalized protein-protein interactionmotif. The p53-binding protein (p53bp) contains fourankyrin-repeat units; its three-dimensional structurereveals the basic features of this motif (top left; Gorinaand Pavletich, 1996). Each repeat contains two shorthelices and an exposed β-hairpin (arrow). Ligandsinteract at two or three points on or between theprotruding hairpin loops. In vitro binding studies haveidentified short (7-25 residue) sequences within atleast five proteins that appear to mediate binding(summarized in Zhang et al., 1998). The structure ofone of these short ankyrin-binding sequences, theankyrin-binding domain in α-Na+/K+ ATPase, isshown. This motif forms an exposed loop on a β-stalk,which is hypothesized to interact with one or twoankyrin repeats as depicted in the cartoon (adaptedwith permission from Zhang et al., 1998).

Fig. 2. Organization of integral membrane proteins by a peripheralskeleton stabilizes biological membranes. Integral proteins canspontaneously distort a bilayer membrane as they seek states oflower energy, causing membrane instability and vesiculation. Thisprocess is ameliorated or controlled if their distribution is managedby a peripheral skeleton.

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important regulatory and structural proteins, includingspectrin. The ARF-induced generation of PtdIns(4,5)P2appears to be a key mechanism by which ARF regulates therecruitment of spectrin to Golgi membranes, whereas reducedPtdIns(4,5)P2 availability (for example, as induced byPtdIns(4,5)P2-chelating agents or inhibitors of PtdIns(4,5)P2synthesis), strongly inhibits the ability of ARF to promotespectrin assembly on the Golgi (Godi et al., 1998).

Beyond stimulating the association of spectrin with isolatedGolgi fractions, ARF dependent increases in PtdIns(4,5)P2levels also stimulate the recruitment to Golgi membranes of adiscrete set of other cytosolic proteins, including ankyrin, actin,a 230-kDa protein, a 170-kDa protein, a 100-kDa protein, anda 30-kDa protein. This set of proteins does not includecoatomer components and is presumably recruited as acomplex with spectrin, given that it dissociates en-bloc fromthe Golgi in the presence of anti-spectrin agents (Godi et al.,1998). Although other interactions between spectrin and theGolgi also exist, including those mediated by adapter proteinsand the broad MAD3 region of spectrin (Y. Ch’ng et al.,unpublished; Devarajan et al., 1997a), the model that emergesis that the synergistic action of at least two binding domains –one constitutive (MAD1), the other ARF regulated (MAD2) –drives the assembly and stabilization of spectrin onto the Golgi.

However, and despite the immunological and functionalsimilarity between the Golgi-associated spectrin and βΙΣ2spectrin, βΙ spectrin transcripts per se have not been detectedin cultured MDCK cells even by sensitive RT-PCRmethodologies (P. R. Stabach, Y. Ch’ng and J. S. Morrow,unpublished). In addition, not all antibodies to βΙ spectrinrecognize the Golgi-associated protein (P. Devarajan and J. S.Morrow, P. Marra and M. A. De Matteis, unpublishedobservations). We therefore originally designated the Golgispectrin as βΙΣ∗ . It has now been identified as βΙΙΙ spectrin(Stankewich et al., 1998). This novel spectrin, whichSakaguchi et al. (1998) independently identified as a ligandfor munc13 (a component of the neurotransmitter-releasemachinery of the presynaptic terminal), cross-reacts with manyantibodies to βΙ spectrin, co-localizes and co-fractionates withGolgi markers, and exerts even more profound effects onsecretory pathway function when transfected into cultured cellsthan do βΙ spectrin peptides (Y. Ch’ng et al., unpublished).However, only a fraction of the total cellular βΙΙΙ spectrin isassociated with the Golgi; the rest is associated with othermembrane fractions (Stankewich et al., 1998). Whether thesubtle differences between the staining patterns obtained withantibodies to βΙ and antibodies to βΙΙΙ spectrin are aconsequence only of differing antibody sensitivities andepitope preferences, or whether other undiscovered spectrinsalso associate with the Golgi, remains an open question. In thisregard, it is noteworthy that two additional spectrins wererecently identified, βIV spectrin (Berghs et al., 1999) and βVspectrin (Stabach and Morrow, 2000; Table 1). These initialreports identify βIV spectrin in association with the nodes ofRanvier and possibly with secretory granules, and βV spectrinas the human orthologue of Drosophila β-heavy spectrin. Inmammalian cells, βV spectrin associates with specializedinternal membrane structures such as the photoreceptor disksof the retinal outer segment (Stabach and Morrow, 2000).Whether these new spectrins play any direct role in the Golgior the secretory or endocytic pathways is unknown.

Finally, a Golgi-associated α-spectrin has not been detected.Whether this is due to poor cross-reactivity of existingantibodies to α-spectrin with a Golgi α-spectrin, or whether allGolgi-associated spectrins are exclusively β type, remains tobe determined. A precedent for the formation of antiparallelhomopolymeric β-spectrin complexes exists: such structuresreside beneath acetylcholine receptor clusters in skeletalmuscle (Bloch and Morrow, 1989; Pumplin, 1995). Yet, thesequence of βΙΙΙ spectrin suggests that it can bind to α-spectrin(Stankewich et al., 1998), and two-hybrid analysis (M. C.Stankewich and J. S. Morrow, unpublished) and the co-immunoprecipitation of βΙΙΙ spectrin with α-spectrin confirmthat the two do associate, at least under some conditions(Cianci et al., 1999a). However, there are currently no specificcandidates for a Golgi-associated α-spectrin.

AnkyrinA second major component of the Golgi spectrin skeleton isthe adapter protein ankyrin. Three ankyrin genes arerecognized (Table 2), and multiple forms arise by alternativemRNA splicing. The Golgi-associated ankyrin isoform,AnkG119, is the only Golgi-associated ankyrin that has beencompletely characterized. Immunofluorescence and cellfractionation reveal that this ANK3family product is tightly co-localized with the Golgi and possibly the ERGIC in MDCKand COS cells (Devarajan et al., 1996; Stankewich et al., 1998),and co-precipitates with Golgi-associated spectrin (Devarajanet al., 1997a, 1998; Godi et al., 1998). Transcripts of otherANK3 family members, including 120-kDa and 100-kDaisoforms, have been identified with late endolysosomes inmacrophages (Hoock et al., 1997). These isoforms differ fromAnkG119in that they lack most of the N-terminal repeat regionbut do have spectrin-binding and regulatory domainscharacteristic of the full-sized ankyrins. Finally, a 195-kDaankyrin that co-localizes with the trans-Golgi (TG) and trans-Golgi network (TGN) has been detected on the basis ofimmunologic criteria (Beck et al., 1997). Although the size ofthis ankyrin is very similar to that of the kidney plasmamembrane AnkG190 (Thevananther et al., 1998), its reactivitytowards antibodies to AnkR and its localization in the Golgisuggest that it is yet another, and possibly unique, form ofankyrin. Collectively, the finding of distinct ankyrins insecretory, endocytic/lysosomal and plasma membranecompartments suggests that this protein family plays a rolein maintaining the distinct membrane profiles of thesecompartments.

ActinA major filament system linking spectrin complexes at theplasma membrane is actin. Although many actin-bindingproteins are associated with the Golgi (Weiner et al., 1993;McCallum et al., 1998; Heimann et al., 1999), the presence ofactin itself on the Golgi has been controversial. Recent in vivoand in vitro studies make the case for Golgi-associated actinand indicate that spectrin provides at least one means by whichactin binds this compartment. Golgi morphology is sensitive toperturbations that disrupt or modify actin (Babia et al., 1999;di Campli et al., 1999). Actin can be co-isolated with Golgimembranes from tissues and cells, and it associates withisolated salt-washed Golgi membranes in vitro (Fath andBurgess, 1993; Godi et al., 1998). Light and electron

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microscopy show that actin filaments are present on the Golgi(Heimann et al., 1999; Valderrama et al., 2000). Theassociation of actin with Golgi membranes in vitro is ATPdependent, stimulated by ARF, and partially inhibited by thesame spectrin-derived polypeptides that interfere with theassembly of spectrin onto the Golgi (Godi et al., 1998). Themolar ratio between actin and spectrin (10:1) on Golgimembranes is higher than that measured at the plasmamembrane (7:1), which suggests that the actin filaments of thespectrin-based skeleton at the Golgi are either longer than thoseat the plasma membrane or that spectrin-independent bindingsites provided by other actin-binding proteins also contribute(Heimann et al., 1999).

Centractin (ARP1)The actin-related protein ARP1 (centractin) binds to Golgi-associated spectrin in vitro, in yeast two-hybrid screens and invivo (Devarajan et al., 1997b; Holleran et al., 1996; E. A.Holleran et al., unpublished). ARP1 is a component ofdynactin, a multimolecular complex required for the dyneinactivity that contributes to intracellular vesicular motility andthe positioning of the Golgi (see below).

AdducinAdditional proteins first identified as part of the erythrocytespectrin skeleton might also contribute to the Golgi skeleton,although at present the available data is incomplete. Adducinis a heterodimeric, calmodulin-, PKC-, and Rho-kinase-regulated actin and spectrin cross-linking protein thatprecipitates with ARP1 and spectrin complexes in culturedcells (Holleran et al., 1996). Immunofluorescence studiesindicate that some anti-adducin antibodies co-localize withGolgi-specific markers in MDCK and COS cells (M. C.Stankewich and J. S. Morrow, unpublished observations) andin NRK cells (M. A. De Matteis and G. Bianchi, unpublished).

THE ROLE OF SPECTRIN IN MEMBRANETRAFFICKING

As noted above, spectrin has been identified on manyintracellular membranes besides the Golgi, includingchromaffin granules, synaptic vesicles, the endoplasmicreticulum, the nuclear membrane, perinuclear Golgi-likevesicles, organelles, and as part of a cytoplasmic meshworklinking unidentified membranous vesicles (Aunis and Bader,1988; De Cesaris et al., 1989; Gregorio et al., 1993; Malchiodi-Albedi et al., 1993; Zagon et al., 1986). These early findingssupport a growing body of recent evidence indicating that,beyond the Golgi, many if not all other compartments of thesecretory and endo/lysosomal pathways (Hoock et al., 1997;Ziemnicka-Kotula et al., 1998) have associated spectrinskeletons. A major issue is now to understand whether theassociation of spectrin with the different intracellularcompartments has any impact on the function of thesecompartments. The evidence so far available indicates that thisis the case, at least for the Golgi-associated spectrin skeleton.This evidence takes several forms.

Along with the identification of targeting domains (MAD1and MAD2) in spectrin, it was observed that transfected βΙspectrin peptides encompassing region-I/MAD1 are potent

dominant negative inhibitors of the transport of certainmembrane proteins to the plasma membrane. In particular,when peptides containing this region were expressed in MDCKcells, Na+/K+-ATPase accumulates in the endoplasmicreticulum, and the glycosylation of its β-chain, which occursin the Golgi, is impaired (Devarajan et al., 1997a). The samerecombinant βΙ spectrin peptides, as well as anti-spectrinantibodies, inhibit the transport of vesicular stomatitis virus(VSV) G protein from the endoplasmic reticulum to the Golgiwhen they are introduced into semi-intact VSV-infected NRKcells. The inhibition of transport of vesicular stomatitis virusG protein (VSV-G) to the Golgi persists even when the peptidesare added after a 15°C temperature block, a manipulation thatcauses protein exiting from the ER to accumulate in theintermediate compartment (Godi et al., 1998). These dataindicate that the spectrin skeleton must exert its role in the earlysecretory pathway, at the ER-to-Golgi interface. Time-lapsevideo microscopy of COS cells transfected with greenfluorescent protein (GFP)-tagged βΙΙΙ spectrin peptidesdemonstrates that spectrin indeed decorates tubular-vesiculartransport intermediates moving along microtubules betweenthe ER and the Golgi, and that spectrin’s region-I/MAD1sequences are required for this movement (Y. Ch’ng et al.,unpublished). These studies also show that ERGIC53 (a markerof the intermediate compartment) and GFP-tagged VSV-Gprotein co-localize with a fraction of βΙΙΙ spectrin in the pre-Golgi compartment in COS cells (Y. Ch’ng et al., unpublished).

Several studies have also documented that ankyrin has afunctional role in the ER exit and proper sorting of manymembrane proteins, including the anion exchanger AE1(Gomez and Morgans, 1993), Na+/K+-ATPase (Devarajan etal., 1997a), Ca-ATPase and the ryanodine receptor (Tuvia etal., 1999), and the lymphocyte tyrosine phosphate phosphataseCD45 (Pradhan and Morrow, 1999). In at least two of thesecases (Na+/K+-ATPase and CD45) this requirement for ankyrinearly in the secretory pathway relates to its ability to link theseintegral proteins to spectrin (P. Devarajan et al., unpublished;Pradhan and Morrow, 1999). Interestingly, although the exit ofanother protein from the ER, E-cadherin, is not ankyrindependent (Devarajan et al., 1997a, 2000), entry of the proteininto the secretory pathway does require β-catenin, a differenttype of putative adapter protein (Chen et al., 1999), which, atthe plasma membrane, links spectrin and actin to E-cadherinvia α-catenin (Nelson and Hammerton, 1989; Provost andRimm, 1999; D. Pradhan et al., unpublished; Roe et al., 1996).Whetherβ-catenin is required to stabilize the conformation ofE-cadherin, as postulated (Chen et al., 1999), or whether β-catenin is needed to link E-cadherin to a spectrin scaffold inthe early secretory pathway – as would be postulated by theSAATS hypothesis (see below) – remains to be determined.Also unanswered are the questions of whether spectrin actuallybinds to any part of the ER, and, if not, where in theintermediate compartment the assembly of a spectrin coat isinitiated.

At present, the evidence suggesting a role for spectrin inpost-Golgi trafficking and in the control of endo/lysosomalcompartments is limited. Specific isoforms of ankyrin (Hoocket al., 1997) and αΙ spectrin (Ziemnicka-Kotula et al., 1998)have been identified in endo/lysosomal compartments; spectrinand ankyrin associate with dynamin and clathrin in vitro andin immunoprecipitates and fractionations of cell lysates (Cianci

M. A. De Matteis and J. S. Morrow

2339Spectrin in the secretory pathway

et al., 1999a; Michaely et al., 1999), and the disruption of theankyrin-clathrin interaction inhibits clathrin-coated-pitbudding in vitro and endocytosis of fluorescent low densitylipoprotein (LDL; Michaely et al., 1999). Finally, othercomponents of the TGN and endocytic machinery, includingannexin VI and amphiphysin, co-immunoprecipitate withβΙΙΙspectrin (M. C. Stankewich, C. D. Cianci and J. S. Morrow,unpublished).

MODELS: THE GOLGI MESH AND THE SAATSHYPOTHESES

Except for its possible inclusion as a Golgi-associated coiled-coil protein (e.g. the golgin proteins, which share an overallorganization reminiscent of spectrin, Kjer-Nielsen et al., 1999),spectrin does not belong to any previously recognized proteinclass implicated in the management of traffic in the secretorypathway. Its pervasive presence in different compartments andthe profound disturbances of transport that accompany itsdisruption suggest that it is central to the function of thesecompartments. Two related models have been proposed asworking hypotheses (Fig. 4).

One model emphasizes the role of spectrin as an organizingand stabilizing force in the Golgi (De Matteis and Morrow,1998; Lorra and Huttner, 1999; Fig. 4A). The existence of aGolgi scaffold or mesh, which by analogy with the plasma-membrane skeleton would act as a template to shape Golgimembranes, is suggested by the presence of a residualdetergent insoluble Golgi-ghost structure containing spectrin,ankyrin, actin and other proteins (golgins) that are collectivelydefined as a Golgi matrix (Beck et al., 1994; Fath et al., 1997;Nakamura et al., 1995). Some components of the Golgi matrixand a matrix-interacting protein, p115, have recently beenshown to regulate the stacking process (Barr et al., 1998;Shorter and Warren, 1999; Shorter et al., 1999). These proteinsact by guiding the alignment and capture of adjacent cisternaein a fashion analogous to the way that they tether transportvesicles to donor/acceptor cisternae. Whether this tetheringmechanism per se is sufficient to shape the Golgi stacks, orwhether it acts in concert with a spectrin-based skeleton,remains to be determined. Also undetermined is whether thereare direct links between the recognized Golgi-matrixcomponents and spectrin. Such an interaction of the Golgimatrix with the ARF- and PtdIns(4,5)P2-sensitive spectrinmachinery might render the entire Golgi skeleton sensitive toG protein and phosphoinositide regulation, and explain therapid and reversible Golgi destacking that accompaniesinactivation of ARF by BFA.

The Golgi scaffold/mesh model also fits well with recentlyproposed versions of the cisternal progression-maturationmodel (Bonfanti et al., 1998; Bannykh and Balch, 1997;Mironov et al., 1997). An essential component of such a modelis a dynamic Golgi scaffold able to undergo rapid remodeling.The regulation of spectrin association with Golgi membranesby ARF and the intrinsic expandability of a spectrin mesh (Fig.1) are both properties that would be important for capturingand remodeling of incoming pre-Golgi intermediates. Thebiochemical maturation of these elements into mature Golgistacks would subsequently rely on the retrograde transport ofGolgi enzymes, whereas the morphological maturation of the

pleomorphic vesicular tubular clusters (VTC) into orderedGolgi cisternae would be sustained by the spectrin skeleton. Inthe later steps of the maturation-progression transport process,dynamic control of the spectrin mesh would facilitate therelease of Golgi membranes into post-Golgi compartments.Within this framework, agents that block association ofspectrin with Golgi membranes are envisioned to act byimpairing the organization and integration of incoming ER-derived membranes into the Golgi complex and thereby inhibitthe transport of cargo molecules from the ER to the Golgi(Devarajan et al., 1997a; Godi et al., 1998).

To account for the role of spectrin in the movement oftransport containers and proteins, and its apparent effects oncargo selection in the secretory pathway, an alternative modelenvisages the presence of a generalized spectrin-ankyrin-adapter protein tethering system (SAATS; Fig. 4B; De Matteisand Morrow, 1998; Devarajan et al., 1997a). This hypotheticalmodel postulates that Golgi spectrin and its associated adapterproteins facilitate the sequestration of integral membraneproteins into transport containers (or else the selective captureof transport containers containing certain proteins) andfacilitates their movement from the ER to the Golgi andbeyond. SAATS probably involves adapter proteins other thanjust ankyrin, and includes members of the protein 4.1, adducin,α-catenin, β-catenin and actin-related protein families. Likeother coat complexes, SAATS assembly on nascent vesicles isregulated by ARF (Godi et al., 1998) and, presumably, SAATSrecognizes complex targeting signals resident in thecytoplasmic domains of integral membrane proteins, such asthose that bind to ankyrin (see Fig. 3). In an alternative view,SAATS can also be conceptualized as a kind of chaperonesystem sensitive to the fidelity of folding of the cytoplasmicdomains of certain cargo proteins in the secretory pathway. Ineither case, a key feature of the SAATS model is the role playedby specific interactions between membrane proteins destinedfor anterograde transport and SAATS. When these interactionsoccur, transport from the ER to the Golgi is efficient; whenthey are blocked, transport is inefficient or blocked altogether.

In contrast to the Golgi mesh hypothesis, in the SAATSmodel spectrin is envisioned to act first on the donorcompartment, clustering and selecting specific molecules orcargo-laden vesicles that have already budded from the ER andthat are destined for anterograde-directed transport. Thiscompartment might be the ER itself, although it is more likelythat SAATS first acts on the VTCs of membranes emergingfrom the ER in the intermediate compartment. This is a crucialsorting station between anterograde-directed cargo moleculesand molecules targeted for retrograde delivery back to the ERdue to the presence of KK motif and phenylalanine residues intheir cytoplasmic domain. This is also the place where vesiclesshed their COPII coats and cluster prior to anterogradetransport along microtubule pathways (Bannykh et al., 1996;Campbell and Schekman, 1997; Lippincott-Schwartz, 1998;Presley et al., 1997; Rowe et al., 1996). It is at this point thatSAATS is most likely to exert its effect, clustering a subsetof the budded vesicles (those that contain sufficientconcentrations of protein or lipids that bind SAATS) into largertransport complexes that are then swiftly moved by spectrin-dynein/dynactin along microtubules to the cis-Golgi. Vesiclesin the intermediate compartment that are not captured bySAATS, or that are extruded from the nascent clusters owing

2340 M. A. De Matteis and J. S. Morrow

Fig. 4. Models of the Golgi-associated Spectrin skeleton.(A) The Golgi spectrinmesh/scaffold. The meshhypothesis attributes to spectrin akey role in organizing andstabilizing the Golgi membranes.In this model, spectrin is a crucialcomponent of the Golgi scaffold,an array of proteins responsiblefor conferring shape andstructural integrity on Golgicisternae. Presumably, spectrininteracts with at least some of theGolgi matrix proteins. Thespectrin-based scaffold isdynamic, undergoing cycles ofassembly (top) or disassembly(bottom). These cycles,controlled by the small GTPaseARF and phosphoinositides,might be localized so as to permitbudding or docking of transportvesicles, or more extended toallow the capture and remodelingof incoming pre-Golgiintermediates into ordered cis-Golgi cisterna and thestabilization of these cisternae asthey mature into medial- andtrans-cisternae. The dynamic andlocalized control of spectrin alsofacilitates the release of Golgimembranes into post-Golgicompartments at the trans pole. (B) The spectrin ankyrin adapter protein tethering system (SAATS). To explain the role of spectrin in both pre-and post-Golgi compartments, as well as the selective effect of spectrin disruption on cargo protein transport, the SAATS model attributes tospectrin a role as both a mechanism for cargo selection, as well as as a tethering link between transport containers and motors. COPII-ladenvesicles bud from the ER and are released into the intermediate compartment (1). Although it is possible that the assembly of SAATS begins asearly as the ER, perhaps in conjunction with the COPII-driven budding reaction (2), it is more likely that SAATS assembly begins in theintermediate compartment and is coincident with the shedding of the COPII coat from newly budded vesicles (1). A key feature of SAATS is itsability to bind selectively and cluster a subset of the budded vesicles (those that contain sufficient concentrations of protein or lipids that bindSAATS) into larger transport complexes that are then swiftly moved by dynein/dynactin along microtubules to the cis-Golgi (3). Vesicles in theintermediate compartment that are not captured by SAATS, or vesicles extruded from the nascent clusters owing to their lack of interaction withSAATS (and that are thereby depleted of proteins tethered by SAATS), presumably assemble COPI coats and are returned to the ER (1), ormove anterograde but less efficiently than those facilitated by SAATS. In the Golgi (3), spectrin is envisioned to continue to function byorganizing protein and lipid mosaics, thereby facilitating their retention and stabilization in the Golgi cisternae, as hypothesized for the Golgimesh. As in the intermediate compartment, proteins not stabilized by SAATS might preferentially enter COPI-mediated retrograde pathways.At the TGN and beyond (4), spectrin-linked mosaics are sorted for delivery to downstream compartments in conjunction with other motor andbudding systems.

2341Spectrin in the secretory pathway

to their lack of interaction with SAATS (e.g. see Fig. 2),presumably assemble COPI coats and are returned to the ER,or move anterograde but at much slower rates than thosefacilitated by SAATS.

Once assembled, it is also envisioned that SAATS mightremain with its associated membrane mosaic as it isincorporated and transported through the Golgi; this processprobably would be accompanied by an exchange of at leastsome SAATS components (e.g. the exchange of AnkG119 forthe 195-kDa ankyrin). As vesicles bud from the TGN, SAATSmight again play a role in directing a subset of cargo-ladenvesicles along microtubule pathways to the plasma membrane(where a final exchange of SAATS components withcomponents of the mature cortical spectrin skeleton, such asthe 195-kDa ankyrin for AnkG190, and βΙΙΙ spectrin for βΙΙspectrin, would occur). Its putative involvement in endosomalrecycling pathways, including those involving clathrin, wouldpresumably follow similar principles. Although there is littlehard data at this point to support the involvement of spectrinor a SAATS-like array in compartments downstream of theGolgi, an interesting prediction of this model is that the sortingof proteins destined for separate membrane compartments (e.g.apical versus basolateral) might begin not in the TGN, as isgenerally envisioned, but as early as the intermediatecompartment, because this would be the site where nascentmembrane- and cytosolic-protein mosaics would be organizedand pre-assembled by SAATS.

Although the two proposed models emphasize differentaspects of spectrin localization and the putative role of spectrinin the secretory pathway, they are not mutually exclusive andclearly merge. Given the increasingly recognized diversity ofthe spectrin gene family, the growing list of recognized adapterproteins and the presence of many alternative transcripts ineach of these families, it is also likely that different isoformsof these proteins operate in each compartment of the secretoryand endosomal pathways. In future work, it will be of interestto test the hypotheses put forth here, and to resolve theinterpretation of these findings in the context of the manylessons learned from the study of secretion in S. cervisiae, anorganism that does not have a spectrin.

Supported in part by grants from Telethon (E732), HumanFrontier Science Program, the Italian National Research Council(99.00127.PF49), and from the National Institutes of Health (JSM).Drs Berghs and Solimend, and other authors who kindly providedinformation in advance of publication for this review are sincerelythanked for their assistance.

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