Unconventional Richard

E. Cheney

Yale University,

myosins

and Mark S. Mooseker

New Haven,

Connecticut,

USA

The unconventional myosins form a large and diverse group of molecular motors. The number of known unconventional myosins is increasing rapidly and in the past year alone two new classes have been identified. Substantial progress has been made towards characterizing the properties and functions of these motor proteins, which have been hypothesized to play fundamental roles in processes such as cell locomotion, phagocytosis and vesicle transport.

Current

Opinion

in Cell Biology,

Introduction

1992, 4:27-35

Ameboid myosins membrane-binding actin-binding site

All myosins contain a conserved - 80.kD ‘head’ domain that can bind to actin, hydrolyze ATP, and translocate along actin Iilaments. Members of the myosin superfamily consist of this ‘generic’ motor domain (and its associated light chains) attached to a variety of unique and functionally specialized tail domains. Previously, the myosins have been divided into two groups - the myosins I and the myosins II - on the basis of whether their heavy chains form monomers or dimers. The myosins II include the conventional two-headed, filament-forming dimeric myosins of skeletal muscle, smooth muscle and virtually all non-muscle cells (reviewed in [ 1,2] ). The term ‘myosin I’ was originally used to identify the unusual monomeric and membrane-associated myosins from Acanfbumoebu, but has come to refer to all single-headed myosins (for reviews see [3*,4*] ). Given the structural diversity of the newly characterized members of this group, we prefer to divide the myosins into two operational categories - conventional and unconventional - with the understanding that the rapidly growing ‘unconventional’ group will eventually include a number of different families or classes. In this review we will divide the unconventional myosins into several different classes that are already apparent and we will summarize recent progress in determining their properties, localization and functions. Special emphasis will be given to the newly discovered Dilute/pI9O/MyO2 class of unconventional myosins, which appears to be associated with vesicle traEicking and is predicted to share features of both myosins I and II. We wtll also discuss a newly recognized sequence motif that is present near the head-tail junction of all myosins and which may play an important regulator)l role as a binding site for calmodulin and other myosin light chains.

I containing both a site and a second

The Acunthamoeba myosin I discovered by Pollard and Kom in 1973 [5] is the prototype of this class of ameboid unconventional myosins (reviewed in [6*] >.The tail domain of this type of myosin has two different functional properties: the ability to bind directly to membranes containing acidic phospholipids and the ability to bind to actin filaments via the presence of a second and ATP-independent actin-binding site. These properties give this class of myosins the potential not only to link membranes to actin and exert force upon them, but also to anchor myosin tails to actin Iilaments, allowing crosslinking and possibly filament sliding. Five very similar myosins I of this type have been identilied, namely Acunthamoebu myosins IA (140 kD), IB (125 kD) and IC (127kD), and Dicpostelium myosins IB (125 kD) and ID [ 30,4*,7,8,9]. The biochemically characterized Acuntbumoeba myosins IA, IE3 and IC are each associated with one or two specific light chains of 17 kD, 27 kD and 14 kD, respectively. Unlike the conventional myosins II, which have large rod-like tail domains consisting almost entirely of coiled-coil a-helix, the tails of these ameboid myosins I are much smaller and are not predicted to form coiled-coil domains capable of dimerization (see Table 1). Furthermore, the tails of these myosins can be divided into three structural or homology domains [3*,4*]. Closest to the head is a region of - 220 amino acids that is rich in basic residues; this region is sometimes referred to as tail homology domain 1 (n-I-1) and has been associated with membrane binding in vitro [ 10,111. Just beyond the membrane-binding domain is a variable

Abbreviations BBMI-brush

border

myosin

@

I; SH-src

Current

Biology

homology

Ltd

domain;

ISSN

TH-tail

0955674

homology

domain. 27

28

Cytoplasm

and

cell

motility

region that is characterized by its unusually high concennation of certain amino acids such as glycine, proline and alanine, and that is called either the GPA domain or tail homology domain II (TI--2). The ameboid myosins I share a third tail homology domain, ‘W-3, equivalent to the - 50 amino acid src homology domain (SH-3 ) which has been identified in a large number of different actinand membrane-associated proteins [3*,9]. The SH-3 domains of the ameboid myosins I are usually located either at the tip of the tail or within the GPA domain, and some combination of the SH-3 domain and the GPA-rich domain is thought to provide the second a&-i-binding site that is present in these myosins. Previous immunofluorescence studies have demonstrated a clear association of the ameboid myosins I with cellular membranes [ 11,121 and suggest that these myosins are involved in vesicle transport [ 131, phagocytosis [14] and ceU movement [ 141. In the ameboid slime mold DicQrxtelium, antibodies to myosins I labeled phagocytic cups and the leading edges of cells in locomotion [ 141, whiIe antibodies to myosin II labeled the posterior half of cells in locomotion and the cleavage furrows of dividing cells. By immunofluorescence and immunogold electron microscopy, Baines and Kom [ 15.1 have now demonstrated a similar distribution of myosin II in Acuw tiumoe6a In addition, using an isoform-specific antibody to Acantbumoeba myosin IC, they showed that this myosin I is closeIy associated with the plasma membrane and the contractile vacuole, but not with other intracellular organeUes. Because previous antibodies to Awnthamoeba myosins IA and II3 stained the plasma membrane but not the contractile vacuole, these results provide evidence for functional specialization among the different ameboid myosins I. Recently, Jung and Hammer [ 161 reported the disappointing result that the deletion of myosin IB from the genetically amenable Dictyosteiium resulted in relatively normal cells that were still able to extend pseudopods, undergo chemotaxis and phagocytose bacteria. In addition to the initially reported reduction in phagocytosis rate [161, a number of more subtle changes in cellular behavior, such as increased pseudopod extension and reduced vesicle movement, have now been documented for these Dic&astefium myosin IB null cells [ 17’1, AIthough it is possible that myosin IB is directly involved in aU of these processes, it is also possible that the absence of myosin IB causes a generalized ‘enfeeblement’ of the ceU that affects many different processes. More striking phenotypes may result from mutants lacking two or more myosin I isoforms if there is some functional overlap among the numerous myosins I known to be present in

Dicpostelium. Several significant results involving myosin I regulation have emerged from studies of the biochemically amenable Acuntbamoeh system in the past year. The ameboid myosins are unusual in that their normally low mechanochemical activity is greatly stimulated by phosphorylation of a conserved region within their head domains. A consensus sequence required for phosphotyIation by this ameboid myosin I head-domain kinase

has now been characterized using synthetic peptide substrates [ 181. The activity of this head-domain kinase is regulated both by autophospholyiation and by acidic phospholipids such as phosphatidyiinositol 1181. The ameboid myosin I kinase, like its substrates, binds to membranes in ritro and is localized on membranes in cells [ 19.1. Using 3 cleverly modified if2 vitro motil ity assay based on a phosphoIipid-coated coverslip. Zot et al. (20’1 demonstrated that membrane-bound Acunttkmoeba myosin IB can move actin filaments, indicating that occupancy of the membrane-binding site does not significantly al?ect the mechanochemical activity of the myosin head. The similar question of whether occupancy of the myosin I membrane-binding site inhibits the set ond actin-binding site as previously suggested ] 111 deserves to be addressed further.

Ameboid myosins membrane-binding actin-binding site

I that contain a domain but lack a second

The closely related Dic(yoste/ium myosins IA ( 113 kD) and IE are similar to the ameboid myosins I discussed above except that they have very short tails that contain the putative TF-I membrane-binding domain, but not the GPA and SH-3 domains thought to be associated with actin binding [4 a,21 1, Thus, these myosins are predicted to lack the nucleotide-independent second actin-binding site of the other ameboid myosins I and are probably incapable of crosslinking actin fiIaments. Unfortunately, almost nothing is known about the localization, biochemical properties, or functions of this interesting group of myosins. (It is important to note that myosin I isoforms from Dictptelium and Acunt~amoeba that have similar names are not necessarily equivaIent isoforms and that two of the Acanthamoebu myosin I genes were initially misnamed with respect to the proteins that they encode

i7l.J

Brush border myosin I: an unconventional myosin from vertebrates that contains a membrane-binding domain and multiple calmodulin light chains Brush border myosin 1, which has been localized precisely as a tether between the plasma membrane of the intestinal microviUus and the undedying bundle of actin liIaments, was the first unconventional myosin to be identified in a vertebrate (reviewed in [22-l). Like Dicpostelium myosins IA and IE, the 119.kD brush border myosin I (BBMI) heavy chain has a relatively short tail that contains a membrane-binding site but appears to lack an actin-binding site. BBMI appears to differ from ameboid myosins I in its complement of multiple caImoduIin light chains, a feature that it shares with several other newly discovered myosins from vertebrates. The tail of

Unconventional

able 1. The myosin

motor

Properties

Conventional Myosins II

and Mooseker

Examples

myosins: Form

dimers

and bipolar

filaments;

responsible for muscle required for cytokinesis capping in Dictyoslelium

Unconventional DilurelplWMy02 Globular

oiled-coil

Skeletal muscle

contraction, and receptor

muscle MII, smooth MII, non-muscle MII,

Dictyostelium (-2OOkD)

MII

myosins: Form

domains

dimers

but

not filaments;

Mouse

Dilute,

the myo2 mutant is defective in budding, has disorganized actin and accumulates vesicles

chicken brain yeast MY02 (18&21S kD)

Form

Acanthamoeba

~190,

a-helix

Ameboid myosins I with tails that bind to membranes and actin

1

Cheney

superfamily. Class

Membrane

myosins

monomers

that

can

crosslink

actin; localized to small vesicles, phagocytic cups, the contractile

binding

vacuole moving

and the leading cells

edges

MIA, MIB, MIC; Dictyostelium of

MIB, MID (125-140 kD)

ATP-independent actin binding

L

Membrane

binding

Ameboid

myosins

I with

predicted

to bind

to membranes

tails

Unknown

Dictyosrelium MIA, MIE t-113

kD)

Brush

border

1 Qi

Membrane

Vertebrate myosins bind to membranes

binding

“II

I that and have

Form monomers; link actin bundles to the plasma membrane

three-four calmodulin light chains

in the microvillus

brain/adrenal (-120kD)

Acanthamoeba hlgh-molecularweight unconventional myosin

Unknown

Acanthamoeba

Ml, Ml

a

high-molecular-weight

I

%

MI

(177 kD) ?

Present

NinaC

in Drosophila

photoreceptor

NinaC

cells; the 174 kD form is localized to the rhabdomere where it may function

splice

forms

(174 and 132 kD)

in phototransduction ?

jchematic diagrams nyosin light chains

of the different classes of myosins by small filled ovals. M, myosin.

are shown

with

the myosin

head domains

indicated

by large open

ovals

and the various

29

30

Cytoplasm

and cell motility

BBMI is extremely basic, although it is now clear that part of the region previously regarded as the ‘tail’ would be better described as a &nod&n-binding ‘neck domain (see below). The BBMI tail does contain a basic domain somewhat similar to the membrane-binding domain of the ameboid myosins I, but lacks both the GPA and SH-3 domains implicated in actin-binding [23,24,25*]. Hayden et al. [25*] have demonstrated that purified BBMI is capable of binding to acidic phospholipids with a & of lOO-3OOnM, and that this activity requires the carboxylterminal - 15kD of the tail. In the future it will be important to determine if the binding of myosins I to phospholipid membranes in vivo is regulated by processes such as phosphoinositide signaling or phosphorylation. A further question that needs to be addressed for BBMI and for membrane-associated myosins in general is whether mechanisms in addition to acidic phospholipid binding are required for targeting these myosins to specific intracellular membranes such as the microvillus or the contractile vacuole. Another basic question is why a molecular motor such as BBMI is used for what appears to be a simple structural role of linking a membrane to actin. A variety of additional roles have been suggested for BBMI, including shedding membranes from the tip of the microvillus, twisting or moving the microvillus itself, moving microvillar membrane proteins, and transporting vesicles. Determination of the precise nature of the interaction between BBMI and the membrane may provide key insights into the actual function of BBMI.

Recent studies of the calcium-dependent regulation of BBMI may provide a general model for the regulation of an emerging family of unconventional myosins that contain multiple calmodulin light chains. Unlike most other calmodulin-binding proteins, BBMI retains its calmod ulin light chains even in the absence of calcium. Currently, there is disagreement over the exact number of calmodulin molecules bound by BBMI, with values of three [26,27*] and four [25*,28*] published recently. Calcium has been shown to have a number of profound effects on BBMI; unfortunately the complexity of these effects is compounded by differences reported among the laboratories investigating this question (discussed in detail in [22*] 1. Taking these results together, however, there is general agreement that calcium, presumably acting through the calrnodulin light chains, has me following effects: first, it alters the accessibility of the heavy chain to proteolytic digestion [29]; second, it activates the magnesium-ATPase activity of BBMI in the absence of actin [26,28*,30]; and third, it inhibits in r&-o motility [ 261. Dissecting the molecular basis of these effects is confounded not only by the presence of three-four bound calmodulins, but also by the key observation that calcium induces the dissociation of one or two calmod ulins from the BBMI [26,28-l. Although these studies show clearly that calcium lowers calmodulin’s alfinity for BBMI, a critical issue to consider is whether or not dissociation would take place in the microvillus, where the calmodulin concentration is estimated to be l-10 millimolar. Consequently, two general models for the regulation of myosin I by calcium and calmodulin have

emerged. The first suggests an on/off regulation of BBMI through a calcium-induced dissociation of calmodulin. The second proposes allosteric regulation of myosin activity through calcium-dependent interactions between light and heavy chains that remain bound to each other. In support of the first model, Collins et al. [26] have shown that the motility of calcium-inactivated BBMI can be restored by the addition of exogenous calrnodulin On the other hand, if excess exogenous calmodulin is present when BBMI is exposed to calcium, a reversible slowing but not cessation of movement is observed, suggesting that regulation through bound calmodulin light chains can occur (Wolenski JS, Forscher P, Mooseker MS: J Cefl Biol [abstract] 1991, 115:27a). Although BBMI was originally reported to be present only in the intestinal brush border, it now appears that there is a family of similar calmodulin-binding unconventional myosins whose members are present in many different vertebrate tissues. Coluccio [31*] has purified and biochemically characterized a 105.kD calmodulin-binding protein from rat kidney brush borders that is similar or identical to intestinal BBMI. In an exciting new discovery, Barylko et al. [32-l purified a lI6-kD calmodulin-binding myosin I from brain and adrenal cortex that by partial protein sequence is distinct from BBMI. Structurally and enzymatically, however, this myosin, which binds to three-four calmodulins, is very similar to BBMI. There is preliminary biochemical evidence for the existence of several additional myosin I-like proteins in vertebrates, including a 13OkD protein from smooth muscle [ 33.1, a IlO-kD protein from macrophages (Atkinson M4 Peterson DM: Biop@ J [abstract] 1991, 59:23Oa), two 130-kD proteins from kidney (Mooseker MS, Heintzelman MB: J Cell Biol [abstract] 1991, 115:331a) and a 153.kD protein from brain (Li D, Chantler PD: Biopkys J [abstract] 1991, 59:229a).

NinaC: highly photoreceptor

divergent cells

myosins

from

fly

The NinaC myosin clearly represents a structurally unique myosin that deserves a class of its own. This myosin was originally identified [34] as the basis of a mutation affecting Drosophila photoreceptor cells. This mutation is associated with abnormal electroretinograms and photoreceptor cell degeneration, The NinuCmyosin occurs in two forms, a 174.kD long form and a 132.kD short form, which differ only in the presence of ahematively spliced tail domains of either 420 or 84 amino acids. The two different tails have little similarity to one another or to any other known proteins. The truly unique feature of the NinuCproteins is the presence of a 30.kD kinaselike ‘nose’ domain appended to the amino terminus of its myosin-like head. Further evidence for the uniqueness of the NinuC proteins comes from inspection of their head-domain sequence (see the alignment in [3*] >, which diverges substantially from all other myosins, even in otherwise highly conserved regions.

Unconventional

Recent localization studies in the fly photoreceptor ceII show that the 174-kD NinaCsplice form is present in the phototransducing organelle known as the rhabdomere, whereas the shorter 132.kD isofonn is IocaIized in the cytoplasm [35**]. The rhabdomere is a microviUus-Iike extension of plasma membrane containing a core of one or two actin fiIaments; the Iight-sensitive rhodopsin proteins are embedded in this specialized plasma membrane domain (reviewed in [ 361). The long NinaCisoform, Iike BBMI, is present in a disposition where it could form the crossbridges observed between the rhabdomere membrane and the underIying actin filaments. The different Iocakzations of the two NinaCsplice forms suggests that the tail of the larger NinaC isoform contains a IocaIization signal for membrane or rhabdomere binding. The function of these intriguing proteins, with a presumed kinase domain riding piggyback on a myosin-Iike motor, is unclear. NinuC could play a role in the shedding of rhabdomere membrane, or it may play a more direct role in phototransduction. In any case, NinaC represents the most divergent member of the myosin superfamiIy yet encountered, and it raises the question of whether other extremely divergent myosins await discovery.

Acanthamoeba high-molecular-weight a new type of unconventional myosin

myosin:

The 177.kD high-molecular-weight unconventional myosin identified recently by Horowitz and Hammer [ 37**] in Acunfbumoebu also appears to represent a new class of myosins. This protein has a myosin-like head domain and an 800.amino-acid tail domain that shares no homology to any of the known ameboid myosins I except for the presence of the 50.amino-acid SH-3 domain at its carboxyI terminus. The tail is not predicted to form a coiledcoil a-helix, and it lacks the putative TH-1 membranebinding domains found in the other ameboid myosin I tail domains. Although little is known about this myosin as yet, it will be extremely interesting to determine its properties and function, as well as whether or not similar proteins are present in other cells.

Dilute, ~1% and MY02 form unconventional myosins

a new class of

One of the most exciting discoveries of the past year was the identification of a new class of unconventional myosins that share some characteristics of both the myosins 1 and the myosins II. The 215.kD gene product of the mouse Dilute coat color locus [38**], a caknodulinbinding protein from brain known as ~190 (EspreaIico EM, Cheney RR, Matteoli M, Nascimento AC, DeCamiIIi PV, Larson RE, Mooseker MS: J Cell Biol [abstract] 1991, 115332a) [39], and the 180.kD product of the My02 gene in yeast [40**] all share certain structural features, including the following: myosin-like head domains that have more in common with one another than with any

mvosins

Chenev and Mooseker

other known myosins; a ‘neck’ region near the head-tail junction, consisting of six tandem repeats of a putative light-chain-binding unit that we caU the IQ motif; a tail domain following these repeats that consists of 150-500 amino acids predicted to form segments of a-helical coiled-coil; and a globular distal tail domain of 400-500 amino acids that is conserved among these three myosins but that has no obvious sequence similarity with the tail domains of other myosins. Because of the presence of the predicted coiled-coil a-helix, one would expect that these myosins form two-headed dimers and would therefore be myosin II-like, but the other structural features of these proteins are clearly unconventional. The phenotypic analysis of these myosins has been particularly interesting. The dilute mutation was named for the ‘dilution’ or lightening of mouse coat colors that it causes. This dilution of hair color appears to be the result of a defect in the transfer of the membrane-bounded and pigment-containing organelles known as melanosomes from melanocytes to the keratinocytes of a growing hair (see [41]). In normal mice the pigment-synthesizing melanocytes have extensive dendritic processes IiIIed throughout with melanosomes; these are somehow engulfed by or transferred to the surrounding keratinocytes. In dilute mice the melanocytes have a normal complement of melanosomes but have very few dendrites. AIthough the classic dilute mutation atfects only coloration because of an unusual tissue-specific effect, many other dilute mutations exist that affect the whole animal [41]. Many of these alleles are deletions that cause neurological deficits such as seizures, and the mice carrying them die within a few weeks of birth, indicating that Dilute is an essential gene. On the other hand, the brains of these mice show no obvious defects in their gross anatomy, and so Dilute itself is apparently not necessary for the extension of neuronai axons or dendrites. Two mutations at different loci, ashen and leaden, have effects on coat color and melanocyte shape that are almost identical to those of dilute. The existence of an additional mutant gene, dilute suppressor, that can suppress aII three coat color mutations [42] suggests that mouse genetics may contribute further to the identification of unconventional myosins and the proteins with which they interact. Mouse brain does contain at least one other protein that shares similarity with the tail domain of Dilute, as a recentIy sequenced cDNA [43*] shows 57% sequence identity with the carboxyl-terminal 700 amino acids of Dilute. The biochemistry of the Dilute/p19O/MyO2 class of unconventional myosins has been explored by studies of the pl9O-caImodulin complex from vertebrate brain. Chicken brain ~190 is a chicken homolog of mouse Dilute, and the two proteins share 91% overall amino acid sequence identity. P190 was originaIIy identified as a calmodulin-binding protein enriched in brain actomyosin preparations [ 391. Although much less abundant than the conventional brain myosin II, ~190 was partially purified as a complex with its EGTA-resistant caknodulin light chains. Work with the ~190 protein also indicates that, Iike the Dilute transcript, it is present in several tissues, but is especially abundant in brain. Immunofluorescence studies of ~190 have revealed a punctate staining pat-

31

32

Cytoplasm

and cell motility

Calmodulin-binding from neuromodulin (29-58) Putative binding chicken (765-909)

region

calmodulinregion from brain ~190

Fig. 1. A putative calmodulin/light chain-binding domain, the IQ motif, is present as one or more tandem repeats in all myosins. The indicated sequences from the myosin neck region were manually aligned with respect to conserved residues within each repeat; these conserved residues are shown in bold. A sequence from bovine brain neuromodulin (top) is also included for comparison. Sequences corresponding to the underlined portion of neuromodulin and the underlined and italicized brush border myosin I splice insert have been shown to bind calmodulin 148-501. The underlined sequence from rat cardiac myosin II is necessary for binding to the essential light chain 1441.

RVAELATLIQKMFRGWCCRKRYO LMRKSQILISAWFRGHMORNRYK QMKRSVLLLaAYARGWKSRR~~~~~~~D~ RRHLAASTISAYWKGYQTRRMYRRY

I

NinaC 174-kD (103~1101)

isoform

Acanthamoeba molecular-weight unconventional myosin (751-779)

high-

Rat cardiac myosin II (780-839)

DKLRAACIRIQKTIRGWLMRKKYM RMRRAAITIQRYVRGHOARCYATFL RRTRAAI I IQKFORMYVVRKRYO CMRDATIALQALLRGYLVRNKYOMM LREHKSI I IQKHVRGWLARVHYH RTLKAIVYLQCCYRRMMAKRELKKL KMHNSIVMIQKKIRAKYYRKOYL DISDAIKYLQNNIKGFIIRORVNDE MKVNCATLLQAAYRGHSIRANVF SVLRTITNL~KKIRKELKQROLKQE HEYNAAVTIQSKVRTFEPRSRFL RTKKDTVVVQSLIRRRAAQRKLKD

Yeast MY02 unconventional myosin (783-926)

Chicken brush border myosin (654-7 16) + alternative p/ice insert

DKAHKAATKIQASFRGHITRKKLKGEKKGD

ELDVKKVIKVQSMMRALLARKRVKGGKVFKLGKK GPEHHDVAASKICIKAFRGFRDRVRLPPLVNEKSG

AVERVTIOIflAGVRRMAFRRLYKRMRAIK

ERLSRIITRIQADARGQLMRIEFKKMVERR DALLVIaMNIRAFMGVKNWPWMKLYFKIKP

tern that is especially prominent in the perinuclear region and at the tips of cellular processes. Most importantly, ~190 binds to actin in an ATP-dependent fashion and has an actinactivated Mg2+ ATPase activity (Espreafico E, Cheney R, Spindola F, Coelho M, Pitta A, Mooseker M, Iarson RJCellBiol [abstract] 199O,lll:167a). In collaborative experiments performed with J Heuser, we have found that highly purilied ~190 molecules are dimers with two rather large heads, a central rod-like segment and two terminal globular domains; ~190 does not appear to form lilaments. The essential My02 gene of the budding yeast .Succ&zromyces cerevkiue was originally identihed in a screen for temperature-sensitive cell-cycle-dependent mutants that fail to bud properly. At the restrictive temperature, myo2 mutants are unable to continue bud growth, exhibit a disorganized actin cytoskeleton, and accumulate vesicles [40**]. This phenotype suggests a defect in the ability to target or transport vesicles to regions of localized cell growth. The distribution of actin in yeast, which have patches of actin in areas of bud growth as well as actin cables extending between the nucleus and the bud, is consistent with this type of role for MY02. The striking finding that a multicopy suppressor of my02 encodes a novel member of the kinesin superfamily of microtubule-based motors (SS Lillie and S Brown, personal communication) also suggests an involvement of My02 in vesicle trafhcking. Furthermore, this result raises the question of the relative roles of microtubule-based versus actomyosin-based systems in vesicle tralficking.

The my02 mutation has also been shown to have genetic interactions (synthetic lethality) with many of the yeast set mutants that aifect post-Go@ steps in the yeast secretory pathway but not with set mutants that affect earlier steps in the secretory pathway (Govindan B, Bowser R, Novick P: J Cell Biof [abstract] 1991, 115185a). Taken together with the phenotype of the mouse dilute mutants and the localization of ~190, all of this work suggests a role for this class of essential myosins in vesicle tralhcking.

A putative

calmodulin/light chain-binding motif is present in one or more tandem repeats in the neck regions of all myosins One of the intriguing findings arising from the sequencing of the dilufe/p19O/MYO2 myosins is the identication in their ‘neck’ regions of six tandem repeats of an extremely basic - 23.amino-acid unit that we refer to as the IQ motif because its conserved core usually fits the consensus IQXXXRGXXXR (see Fig. 1). This unit is particularly interesting because it is a previously unrecognized motif that is present in one or more copies in the neck regions of all myosins. The conventional myosins II contain one well conserved repeat and a second less conserved repeat in areas implicated in the binding of their essential and regulatory light chains, respectively [ 44-461. Both light chains are known to be members of

Unconventional

the calmodulin/EF-hand superfamily of proteins [47]. We note that all of the unconventional myosins also contain at least one IQ motif. For example, the Acunfbumoeba highmolecular-weight unconventional myosin appears to contain only one repeat, whereas the long and the short forms of NinuCeach have two repeats, despite the presence of the splice junction within the second repeat. As pointed out by Mercer et al. [38-l, BBMI, which binds three or four calmodulin light chains in the absence of calcium, has three previously unrecognized repeats in the region thought to be responsible for calmodulin-binding. A fourth IQ repeat would be formed by the 29 amino acids added to this region in a recently identified alternative splice; the synthetic peptide corresponding to this insert was also reported to bind to calmodulin in the absence of calcium [48]. P190, which has six IQ repeats, was originally studied as a calmodulin-binding protein and is isolated in the absence of calcium with multiple calmodulin light chains. The IQ motif shares sequence similarity to the calmodulin-binding domains of non-myosin proteins (Cheney RE, Espreaiico EM, Larson RE, Mooseker MS: J CefI Biol [abstract] 1991, 115:432a), such as neuromodulin, a well studied 24.kD neuronal protein that also binds to calrnodulin in the absence of calcium [49,50]. We suggest that IQ motifs provide binding sites for proteins of the calmodulin/EF-hand superfamily and that these sites generally retain their binding activity even in the absence of calcium. What the actual structure of the IQ motif is, how it might distinguish between different types of light chains, and exactly how it is Involved in myosin regulation are unclear. It is clear, however, that the neck region of the myosin head is subject to a surprisingly large amount of structural variation, containing from one to six of these repeats in different myosins.

Chenev and Mooseker

mvosins

115:331a). With this potentially very large pool of unconventional myosins the question of what they are aU doing naturally arises. Although it is possible that each type of motor associates with only one organelle and has one specific function, it is also possible that processes such as cell locomotion involve dozens of motors where any one motor may only contribute to a cell-biologically slight, but evolutionarily significant, increase in efficiency. In either case, the purification and localization of unconventional myosins such as NinuC, ~190 and the Acuntbumoeba high-molecular-weight myosin should lead to rapid progress in determining the properties of these proteins, and analysis of unconventional myosin function in genetically amenable systems such as yeast, DicQastelium and Drosophila may allow the identification of specific biological actions for each motor.

Acknowledgements We wish to thank our colleagues in the unconventional myosin for sharing their unpublished data, manuscripts and abstracts. note that the 1991 Journctl o/Cell Bio/oa abstract supplement tains many additional abstracts invoking myosin 1.

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and recommended

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WARRICK

Cell 2.

HM.

SPLIDICH JA

Motility.

KORI‘;

ED,

reading

within

Myosin

the annual

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RetI Cell Biol

Arznu

field We con-

HAMMER JA Myosins of 1988, 17:23-45.

1987.

and

period

of re-

Function

in

3:379-421.

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Cells.

Annu

Ret! Biopkys G5etn

Perspectives The number of unconventional myosins being discovered currently is almost frighteningly large. In addition to the unconventional myosin ~!4Y02, yeast contain an ameboid myosin-I like protein (MYO-?) (Goodson HV, Titus M, Spudich JAJCell Biol [abstract] 1990,111:168a) and a novel, six-IQ-repeat myosin (MyO4) (SH Lillie and SS Brown, personal communication); thus, three out of the four known myosins in yeast are unconventional myosins. In Dictyafelium, which has a single myosin II, there is preliminary evidence for at least nine unconventional myosins [ 511. In Drosophila, which has two conventional myosin genes, two unconventional myosins in addition to NinuC have been identified recently (Kellerman K et al.: J Cell Biof [abstract] 1990, 111:169a; D Kiehart, personal communication). In vertebrates, where for many years BBMI was the only known unconventional myosin, we may be poised for a vimial explosion of unconventional myosins. In brain alone, where Dilute/p190 is already found, there is prekminaty sequence evidence for three additional unconventional myosins similar to BBMI (Sherr EH, Greene IA J Cell Biol [abstract] 1991,

TD, DORERSI’EIN SK, ZOT HG: Myosin-I. Annu Reu 1991, 53:653681. A recent and comprehensive rwiew of the unconventional myosins which also includes an alignment of myosin head sequences showing putative myosin I consensus regions.

3.

Pow

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Physiol

-1. H,MMER JA Novel Myoslns. Trends . I :5ct56. A concise review of the unconventional myosins with of myosin I tail structure.

5.

Pow from

TD,

KOKN

Acuntbumoebu Muscle Myosin. J

KORN

ED:

.

ture.

Cut-r

7.

review

summarizing

JUNG C, ~CHMILIT

JLING

Biol them

a useful

Myosinz

summary

1. Isolation

of an Enzyme 1973,

1991,

Similar

to

248:46824690.

Myosin I: Past, Present, and Fu1991. 38:1330. work with the Acanfbamoeba myosins I.

Top Membr

CJ, HAMSTER JA: Myosin

of Acunthumoebu and Evidence for 1989, 82:26%280 8.

Acunthamoeba

custellunii

Acunthumoeba

6. A recent

ED:

Cell Bid

custelluni: the Existence

I Heavy-chain Cloning of a Second of a Third Isofortn.

Genes Gene Gene

HAVMER JA: The Heavy Chain of AcunII3 is a Fusion of Myosin-like and NonSequences. Pra: Null Acud Sci U S A 1987,

G. KORN

thumoebu

ED,

Myosin

myosin-like

84:672&6724. 9.

JUNG G. SAXE CL

coideum Chain.

Contains

KIMMEL AR, HA~SIER

a Gene

JA: Dlctyostelirrm

Encoding

Proc Nat1 Acad Sci II S A

1989,

a Myosin 86:6186-6190.

disI Heavy

33

34

Cytoplasm

and cell motility

. 10. 11.

ADAMS RI. Pouiuu, TD: Binding Lipids. ic;arure 1989, 340:56%%8. H, BOWERS B, KORN sociation of Acantbamoeba 109:151+1528.

MIYATA

of Mvosin ’ ED:

Myosin

12.

GAD~SI H, KORN ED: Evidence for the Iar LocaIization of the Acantbamoeba Nature 1980, 286:452456.

13.

ADAMS RJ, POUARD TD: Propulsion kom Acantbamoeba Along Actin Nature 1986, 322:754-756.

14.

FlJKUl

Y,

cated

at the

LYNCH

Amoebae.

15. .

Nature

BAINES IC, KORN

Plasma Membrane I. J Cell Biol Differential Myosin

1989, ED:

As1989,

IntraceIIuIsoenzymes.

of Myosin

IC

and

II in Acantbamoeba castellani by Indirect rescence and Immunogold Electron Microscopy. 1990, 111:189~1904.

Myosin

ImmunofluoJ Cell Biol

JUNG G, HAMMER JA: Generation

and Characterization fyosrelium Cells Deficient in a Myosin I Heavy form. J Cell Biol 1990, 110:1955-1964.

17.

WE%EIS

D, MURRAY J, JUNG

.

El Null Motility.

Mutants of Dictyostelium CeLl Motil Cyakeleton

G. HAMMER JA, Sou

of DicChain IsoDR

Exhibit Abnormalities 1991, 20:301-315. approach, Diqwfelium

19.

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BRZESKA H, LYNCH TJ, MARTIN B, CORKXANO-MURPHY ED: Substrate Specificity of Acantbamoeba Myosin

Chain

Kinase

cbem

1990,

as Determined

with

Peptides.

H,

ED:

D, BAINES IC. BRZFXA

KORN

localization of Myosin I Heavy Chain Kinase to Plasma Membranes. J Cell Biol 1991, 115:1w119. paper demonstrates that the Acanihm& heavy-chain

I Heavy J Biol

ImmunoIsolated

SK, POUARD TD: Myosin-I Moves Actin on a Phospholipid Substrate: Implications for Targeting. J Cell Biol 1992, 116:367-376. demonstration that Acantbamoeh myosin IB bound to

Filaments Membrane

21.

TITUS M&

tor 22. .

WA~RICK

Genes

in vitro

HOSH~MARU

Mammalian Biol Ckm

Brush

was made posmotility assay invoking

Multiple Cell Reguhtion

border

and

Border

Myosin

A&n-based 1989,

1987,

I. Cutr

myosin I with a derailed regulation by calcium.

M, NAKANISHI S: Identification Myosin Heavy Chain by

Mo1:55-63.

29.

of

of a New Type Molecular Cloning.

of J

262:1462>14632.

of the IlO-kD-CalmoduIin MicroviIIus Shows that

Complex of the this Mechanoenzyme

COLUNS

Cefl

Bid

1989,

MS: BInding

JS, MOOSEKER

I to PhosphoIIpid

of Brush J Cell Biol 1990,

Vesicles.

K,

SEUERS JR,

P: CaImoduIIn DiiiMyosin I (IlO-kD-CalmodIn Vitro. J Cell Bill 1990,

MAT~UDAIRA

Border Activity

Il.

H, COUINS JH: Ca+Z Stimulates tbe Mg’ 2-ATPase Activity of Brush Border Myosin I with Three or Four Cahnodulin Light Chains but Inhibits with Less than Two Bound. / Biol @em 1991, 266:1312-1319.

SWANLJUNG-COUINS

COLUCCIO IM. BRETXHER A: Iar llOK-CaImoduIin Complex

Mapping

of

(Brush

Border

of Fragments Containing Sites and Demonstration Conformational Change.

the

MicrovUMyosin

I):

the

Catalytic and of a Calcium Biocbemkfry 1990,

30.

CONZEL~!AN KA, M~~SEKER MS: The IlO-kD Protein-calmodulin Complex of the Intestinal MicrovilIus Is an Actin-activated MgATF’ase. J Cdl Biol 1987, 105:31%324.

31. .

COLUCCIO,

LM: Identification of the Microvillar 1lOkDa Calmodulin Complex (Myosin-1) in Kidney. Eur J Cell Biol 1991. 56:286-294.

Avian

similar or identical fo intestinal BBMl was tubt!!e brush borders and biochemically

BARYIKO B, WAGNER MC, REIZES 0, ALBANESI JP: Putication and Characterization of a Mammalian Myosin I. fmc N&l Acad Sci USA 1992, in press. A novel 116.kD myosin I from brain and adrenal tissue was purified and characterized AIthough clearly distinct from BBMI, this new myosin is similar to it in many respects. including its tendency to bind to three-four calmodulins in the absence of calcium.

32.

33. .

KOHAMA

K, LIN Y, TAKANO-OHMLIRU

Like Protein (suppl):59-61.

from

Smooth

Reports the purification of a 130.kD can bind to actin. has actin.activated to lack light chains.

35. . .

MONI’EU

PORTER

H, ISHIKAWA

Muscle.

j

myosin from Mg-ATPase

Cell chicken activity,

R: A Myosin-

Sci

1991,

14

gizzard that and appears

C, RUBY GM: The Drosophila ninaC Locus EnTwo Photoreceptor CeU Specific Proteins With DoHomologous to Protein Kinases and tbe Myosin Chain Head. Cell 1988. 52~757-772. J. HICKS

JL,

WILUAMS

DS.

Momu.

Localizations and Requirements for the ninaC Kinase/Myosins in Photoreceptor

C: DUTerentiaI Two Drosopbflu CelIs. J Cell Rio1

1992, in press. By immune-electron microscopy, the 174&D NinuC isoform was shown to be present in the rhabdomere, while the 132.kD isoform was present in the cytoplasm. The 174.kD but not the 132.kD isoform was shown to be necessary for normal photoreceptor function, and a direct role for the 174.kD protein in phototransduction was suggested.

36. is

protein proximal

.

codes mains Heavy

Top Membr discussion

Myosin

Identification F-actin-binding Ion Dependent

34.

GARCLA A, COUDRIER E, CARBONI J, ANDERSON J, VANDEKERKHOVE J, M~~~EKER M, LOWARD D. ARPIN M: Partial Deduced Se-

quence Intestinal

J

BBMl was reported to bind four caImodulins in the absence of calcium, whereas the presence of micromolar concentrations of calcium caused the dissociation of two calmodulins, leading to an inhibition of ATPase activity.

MOOSEKER MS, WOLENSKI JS, COLE~MN TR, HAYDEN SM. CHEW RE, ESPREAFICO EM, HE~IMAN MB, PETERSON MD: SwcturaI and Functional Dissection of a Membrane-bound

A recent review of brush this protein’s biochemistry

24.

tilaments

HM, SPUDICH JA

in Dictyostelium

Mechanoenzyme: 1991, 3B:31-55.

23.

actin

SM, WO~ENSKI

Border

A calmodulin.binding purified from kidney characterized.

ZOT HG, DOBE&TE~

This formal membranes is still capable of moving sible by the development of a mc&ied a phospholipid-coated coverslip.

Family.

29:1108%11094.

myoability

This kinase. like its myosin I substrates, can bind to purified plasma membranes, and that much of the kinase is IocaIized to membranes in cells. It also suggests that autophosphorylation of the kinase may regulate its association with the membrane.

20. .

28. .

A. KORN

Synthetic

HAVEN

and myosins

in

265:1613%16144.

KUIESU-LIPKA

I

27. COUNS K, MArSUDAIR.4 P: Differential Regulation of Verte. brate Myosin I and II. J Cell Sci 1991, 14 (suppI):ll-16. A brief review that discusses tierences in the reguhtion of myosins I

Myosin

By use of a sophisticated image anaIysis sin I8 null cells were shown to have subde deficiencies in their to extend pseudopods, phagocytose and transpon vesicles. 18.

Myosin

ation Regulates Brush ulin) Mechanochemical 110:1137-l 147.

Myosin I is LoDictyostelium

Using an isoform-spec&c antibody in immunoIiuorescenceand immune. electron microscopy, Dicfp&ium myosin IC was shown to be localized to the contractile vacuole and to the plasma membrane, which is in conuast to the previously reported sraining patterns for the IA and IB &forms. The Acuntbamoeba myosin Il was localized to the cleavage furrow of dividing ceUs and the rearward end of crawling cells.

16.

25. .

26.

341:328-331.

Localization

the

111443451. BBMl was shown to bind with high affinity to liposomes composed of anionic but not neutral phosph0Iipid.s; this membrane-binding activity appears fo depend on the carboxy-terminal 15 kD of the heavy chain. The primary structure of the tail is aLso discussed with respect to putative membraneand cabnodulin-binding regions.

of OrganeIIes Isolated Filaments by Myosin-I.

ED: of Locomoting

Edges

of

109:289+2903.

TJ, BRZESKA H, KORN

Leading

a Member

I to Membrane

WIWS Turnover.

DS: Actin BioEsqs

Filaments 1991,

and Photoreceptor 13:171-178.

Membrane

Unconventional 37.

HOROWTIZ

JA, HAMMER JA

.. Heavy Chain. J Biol Own Reports the sequence of a 177kD tional myosin from Acanthamoeba not predicted to form a coiled-coil to any known myosin. The protein sence but not the presence of ATP of unconventional myosin distinct 38.

..

A New Acunthamoeba Myosin 1990. 265:2064620652. high.molecular-weight unconventhat has a large tail domain which is a-helix and that has little homology appears to bind F-actin in the aband probably represents a new class from the other ameboid myosins 1.

M!ZRCER JA SEPERACK PK, STROBEL MC, COPELAM) NC, JENKINS NA Novel Myosin Heavy Chain Encoded by Murine dflute

Coat Color Locus. Nature 1991, 349:709-713; 352:547. This paper shows that the gene responsible for the classic mouse hair color mutation known as diluteencodes a novel type of unconventional myosin. This myosin’s predicted structure as well as the distribution and processing of its mRNA are discussed.

39.

40. ..

LWSON RE, ESPINW~A FS. ESPREAFICO EM: Cahnoduhn-binding Proteins and Calcium/CahnoduIin-regulated Enzyme Activities Associated with Brain Actomyosin. J Neurocbem 1990. 5412881294. JOHNSTON

GC,

PRENDERGAST

J&

SINGER

RA:

The

Sac&a-

romyces cereuisiae MY02 Gene Encodes an Essential Myosin for Vectorial Transport of Vesicles. J Cell Biol 1991. 113:539-551. This paper reports the sequence of an unconventional myosin from yeast that is stmcturally similar to the mouse Dilute myosin. Phenotypic analysis of cells with the temperature-sensitive my02 mutation shows that these cells accumulate vesicles, have a disorganized actin cytoskeleton, and are unable to form buds properly. 41.

SILVERS WK: Ruby-eye-2.

Dilute and Leaden, the p-Locus, In GXI Colors of Mice: a MoaW

Gene Acfion and Inferaction New York: Springer Verlag

42.

edited 1979, pp

M@XE KJ, SWUNG DA, COPE~AND of the Murine dilute suppressor Coat Color Mutations. Genetics

Ruby-eye,

NG. JENKINS NA interaction Gene (dsu) with Fourteen 1990,

125:421-430.

REED-F•

URQUU

Chenev

L, WV

and Mooseker

43. .

HUNG Cloning

E. Wu

JY:

Molecuiar

44.

MCNALLEY EM, BRAVO-ZEHNDER MM, IEINWAND IA: Identilication of Sequences Necessary for the Association of Cardiac Myosin Subunits. J Cell Biol 1991, 113:585590.

45.

MITCHELL EJ, KARN J, BROWN KEN~RICK~~ONES J: Regulatory and ing Sites in Myosin Heavy Chain Site-directed Mutagenesis. J Mel

46.

NAY L, GOODWIN EB, SZE.NI”-GYORGYI AG: mary Structure of a Scallop Striated Muscle Chain. J Biol Cbem 1991, 2661846~18476.

47.

KRITSINGIZR RH: Structure lated Proteins. CRC Cril

and Amino Acid Sequence of Brain L-Glutamate Decarhoxylase. Proc Natl Acud Sci (I S A 1990,87:84918495. The mouse brain cDNA sequence reported in this paper shares no homology to any of the other known glutamate decarboxylases but does share 57% identity with the tail domain of the mouse Dilute myo sin. This indicates that mouse brain contains at least one additional protein that is structurally similar to the Dilute tail domain.

48.

HALSAU DJ. HAMhm

JA

and

49.

Wwi

BT,

ALEXANDER

DR,

WAISH

KA: Amino Calmodulin

rospecific

DM, Nm A, JAKES R, Essential Light-chain-bmdSubfragmentMapped by Biol 1989, 208:19%205.

Evolution

Rev Biocbem

Isoform a 29Residue FEBS

KA,

Sequence Protein.

8:119-174.

of Chicken Brush Inserted Sequence

Lett 1990,

MASURE HR,

Acid Biding

Complete PriMyosin Heavy

of Calcium-modu1980,

A Second

Border Myosin I Contains that Binds to Calmoduiin.

267:12&130.

CIMLJXR BM, of P-57, Biccbemisty

STORM a

Neu-

1987.

26:746X&7466. 50.

CHAPMAN

ER, ALI D, ALEXANDER

KA, NICOLX)N TA, STORM DR: of the Cahnoduiin BindIng Domain of Neu-

Characterization

and

for Mammalian by Silvers WK [book]. 83900; 104-107.

WM,

mvosins

romodulin.

51.

J Biol

&em

1991,

HAMMER JA, JUNG G: Molecular Chain Genes. J Ceil Sci 1991,

266:207-213.

Cloning of Myosin 14 (suppl):3740.

RE Chewy and MS Mooseker, Department of Biology, PO Box 6666, New Haven, Connecticut 06511, U S A

I Heavy

Yale University,

35

Unconventional myosins.

The unconventional myosins form a large and diverse group of molecular motors. The number of known unconventional myosins is increasing rapidly and in...
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