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Transworld Research Network37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India

Recent Res. Devel. Cell Biol., 1(2003): ISBN: 81-7895-116-9

9

Analysis and function of Rab

GTPases in Dictyostelium discoideum:

Key regulators of vesicular

membrane transport and

multicellular development

David B. Stephens, Biswa N. Mitra and John M. Bush

Department of Biology, The University of Arkansas at Little Rock, Little Rock, AR

USA 72204

AbstractThe nature and functions of the Rab small

GTPases in the slime mold Dictyostelium discoideum

are reviewed. Rab proteins have been identified as

being involved in vesicular traffic, and the organism

Dictyostelium discoideum is a well-characterized

model system for the study of eukaryotic cytology.

This review attempts to describe the intersection pointof these two areas of study.

IntroductionRab proteins are part of the Ras superfamily of

small GTPases, specific cellular enzymes that bind

either GTP or GDP nucleotides.[1, 2] They undergo a

cycle modulated by co-factor proteins which influence

Correspondence/Reprint request: Dr. David B. Stephens, Department of Biology, The University of Arkansas at Little Rock

Little Rock, AR, USA 72204. E-mail : [email protected]

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David B. Stephens et al.2

the hydrolysis rate of bound GTP to GDP, release of GDP, and binding of new GTP.[3,

4] Rab proteins bound to GTP are said to be in their active state, while those bound to

GDP are said to be inactive. All reported Rab GTPases are thought to function in the

processes of eukaryotic vesicular transport between intracellular compartments,

endocytosis, or exocytosis.[5-8] Over 30 mammalian Rab proteins have so far been

described, and at least 11 in yeast.[9-11] Rabs have been shown to be targets for

pathogens.[12] The levels and activities of many Rab proteins are known to be

responsive to cell cycle and differentiation cues.[13, 14]

Dictyostelium discoidium (Dd) is a soil-living amoeba that feeds on bacteria, an

important component of the detritus layer of forest ecosystems. It is a facile system forbasic biomedical research in cell and developmental biology, having unique advantages for

studying fundamental cellular processes with powerful molecular genetic tools, processes

either absent or less accessible in other organisms.[15-19] It is a haploid organism, with an

estimated number of genes of from 8,000 to 10,000; many of the known genes show a high

degree of sequence similarity to genes in vertebrate species.[20] Some of the processes

shared byDdand more complex organisms include:

1. Cytokinesis useful for developing models in immunology, tissue

maintenance, and cancer.

2. Cell motility useful for developing models of tumor metastasis and

endothelial angiogenesis.

3. Phagocytosis useful for developing models of phenomena such as immune

surveillance and antigen presentation.

4. Chemotaxis and signal transduction useful for developing models ofprocesses such as inflammation, arthritis, asthma, lymphocyte trafficking, and

axon growth.

5. Developmental aspects - cell sorting, pattern formation, and cell-type

determination, features of embryogenesis and neoplastic transformation.

The National Institute of Health has thus designated Dd as a model organism for

functional analysis of sequenced genes.[21]

The process of vesicular transport has been extensively studied in many systems

including the simple eukaryote Dd.[22, 23] Ddshares similarity with human macrophage

cells in the presence of a prominent endocytic system and a highly phagocytic nature.[24]

Earlier studies have revealed that extracellular material is endocytosed via clathrin coated

vesicles and transported through endosomes into acidic lysosome-like vesicles in all

eukaryotic organisms including Dd. Uniquely in Dd, cargo is then transported from

lysosomes to larger neutral post-lysosome compartments prior to egestion. The Ddendocytic pathway also differs from mammalian cells in the lack of vesicular recycling

from endosomal compartments back to the cell surface; instead, endocytic vesicular

trafficking is linear and extracellular cargo moves through all of the endocytic

compartments before leaving the cell.[25-27]

Structure of Rab proteinsRab proteins, as part of the Ras superfamily of small GTPases, are typified by a

highly conserved three-dimensional crystal structure in the guanine nucleotide binding

domain, consisting of a central six-stranded sheet enclosed by 5 helices.

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Rabs inDictyostelium discoideum 3

Figure 1. Structure of p21 Ras.[28]

Figure 2. Topological Structure of Ras Superfamily[29]

Ras superfamily proteins have been found to be characterized by a number of structural

features:

A. Guanine nucleotide binding domain.

B. Effector interaction regions.

C. Hypervariable C-terminus.

A. Guanine nucleotide binding domain

The bound nucleotide is contained in a pocket composed primarily of loops L1, L2,L4, L8, and L10.[29] Conserved sequence elements in this domain includes the

following guanine (G) binding and phosphate/Mg2+ (P/M) binding components:

P/M 1, also called the P-loop: This element has the Ras superfamily consensus

sequence GxxxxGK S/T, and extends from L1 into -helix 1. Forms a loop where

the backbone amide hydrogens and the -amino group of the conserved lysine

interact with the charged - and -phosphate groups of bound guanosinepolyphosphate. The conserved serine/threonine residue coordinates the Mg2+

cofactor necessary for GTP/GDP hydrolysis; substitution with alanine or asperagine

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severely modifies interactions between protein, metal, and nucleotide, thereby

producing a deactivated mutant, with the protein locked into the GDP-bound

state.[30-32]

G1: A conserved phenylalanine/tyrosine residue situated in L2, important in forming a

hydrophobic environment for the bound guanine. Mutation of this residue to leucine

increases dissociation of bound guanine and thereby weakens guanosine

polyphosphate binding by 140-fold.[32, 33]

P/M 2: A highly conserved threonine also located in L2 and necessary for Mg2+

coordination to the - and -phosphate groups of GTP is referred to as P/M 2. This

threonine is also part of the Switch I region that along with the glycine of P/M 3 inthe Switch II region - forms part of the conformational response mechanism to GTP

hydrolysis. Mutations in the conserved threonine and P/M 3 aspartate residues fail

to be activated by GAP, perhaps due to incorrect metal coordination.[31, 32]

P/M 3: P/M 3 is located from -strand 3 into L4, and has the Ras superfamilyconsensus sequence DxxG Q/T E; in Rab this sequence seems to be even more

tightly conserved as WDTAGQ. Occurs at the beginning of the Switch II region.

The aspartate in this motif is in the second coordination sphere of the metal and

hydrogen bonds to other coordinated species and is involved with stabilizing an H 2O

molecule coordinated to Mg2+. A mutation of the conserved glutamine to leucine

cannot activate water as a nucleophile - a Rab with such a mutation is incapable of

hydrolyzing bound GTP, rendering it an activated mutant in the GTP-bound

state.[34]

G2: Ras superfamily consensus sequence N/T K/Q xD, Rab conserved motif GNKSDlocated from 5 into L8. Mutation of the conserved asperagine to isoleucine greatlyincreases the nucleotide dissociation rate and produces a deactivated mutant, with

the protein in an nucleotide unbound state.

G3: Ras superfamily consensus sequence F/Y xExSA K/L, located from 6 into L10.The lysine in this motif seems to function to stabilize the phenylalanine in G1 by

hydrophobic interaction, and the serine forms a hydrogen bond with the G2

aspartate residue. The main chain nitrogen of the alanine in this motif hydrogen

bonds with O6 of the bound guanine, perhaps aiding in the base specificity of this

binding pocket.[35, 36]

B. Effector interaction regionsThree structural domains have been proposed to specify the stage in cellular

transport at which members of the Rab family act: the switch domains, the C-terminalhypervariable region, and a region corresponding to 3-L7 in the structure of p21.[37]

The switch domains and 3-L7 have been shown determinant in specific GEFinteractions.[38] Rab5/Rab6 chimeras have been used to demonstrate the sufficiency of

the C-terminal hypervariable region for organelle targeting, but the necessity of the N-

terminus, 2-L5 (switch II), and 3-L7 for full functional interchange.[39] Othermembers of the Ras superfamily are known to vary from the structure of p21, mainly by

N- and C-terminal extensions and loop insertions, especially L7 and L9.[36] These

structural features seem to be conserved among various Rabs between species.[40, 41]

The identity of the switch domains was originally developed using p21 Ras

crystallographic data, using Ras bound to GDP and to nonhydrolyzable GTP analogs;

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this model was then expanded by sequence-comparison to encompass the entire

superfamily. Crystallographic data indicates the existence of two allosteric switch

regions, regions that show large amounts of conformational change based upon binding

to GTP versus GDP, and these regions are proposed to be contact domains for various

effectors.[42] The Switch I (SWI) region corresponds to most of the residues in L2, and

Switch II (SWII) corresponds to parts of L4 and 2.[29, 43] Some typical switchdomain sequences are shown in Table I; overlap between switch and nucleotide binding

domains are shown in boldface, with the bold threonine in SW1 the P/M 2 residue and

the bold triplet at the beginning of SWII is the end of P/M 3.

Table I

The switch domains have been shown to have an essential role in the binding of a

number of accessory factors, including REP,[44]

The C-terminal hypervariable region of Rab proteins has been implicated in intracellular

compartment specificity. [37, 45] Studies with chimeric Rab5/Rab7 have shown that the

terminal 35 amino acids are sufficient to switch targeting.[45] In studies with Rab6, the

C-terminal hypervariable domain was shown to be not essential for Golgi complex

targeting but is required to prevent prenylated and palmitoylated Rab6 from localizing to

the plasma membrane.[46] On the other hand, experiments using Ras/Rab chimeras

showed that there are sequences in the N-terminal 71 amino acids of Rab6 which are

required for Golgi complex localization and show that these sequences comprise or

include the effector domain.[46]

Rabs have been shown to be substrates for prenylation by the enzyme Rab

geranylgeranyl transferase, with prenylation motifs distinct from other members of the

Ras superfamily.[46-49] Double prenylation modification of Rab is not only essential

for membrane insertion, but also adds specific prenylation to the already knowndeterminants of Rab localization. [50, 51] The enzyme geranylgeranyltransferase II

transfers the geranylgeranyl group from the pyrophosphate (see Figure 3) to cysteine

residues at the C-terminus of Rab (vide infra).

O P

O

O

O-

P

O

O-

O-

Figure 3. Geranylgeranyl pyrophosphate

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A somewhat unorthodox approach was reported categorizing 100 constitutively-

active mutant Ras superfamily GTPases by resultant cell morphologies, then searching

for relevant residues by calculating conservation within each category versus divergence

between categories for each amino acid.[52] Multiple mutations, some at sites outside

of known interaction domains, were ranked for predictive purposes and then

demonstrated by switch-of-function experiments. The use of cell morphology as a basis

for classification could implicate cytoskeletal involvement at these sites, but this work

has not yet been reported.

Another avenue for the generation of functional heterogeneity is the production of

isoforms by alternate mRNA splicing.[53, 54] Sequence comparisons have led tophylogenetic categorization of mammalian Rabs into a number of gene subfamilies: [55,

56]

Rab1a/b and Rab35

Rab3a/b/c/d

Rab4a/b and Rab14

Rab5a/b/c

Rab6a/b

Rab 8a/b and Rab10

Rab11a/b and Rab25

Rab22a/b

Rab27a/b

Rab32 and Rab38Rab40a/b.

Apparently the result of gene duplications, these subfamily members have diverged

in function.

Cellular localization of Rab proteinsAn early clue as to the function of Rab proteins came from their intracellular

localization at any single time-point, a large proportion of the Rab proteins present are

membrane-bound. Further investigation showed that different Rab proteins

characteristically associate with different intracellular compartments, which contributed

to the growing hypothesis that Rab proteins function in intracellular transport processes,

as did the observation that specialized Rabs are produced in cells differentiated to

unusual membrane-traffic requirements.[57-64] The intracellular localization ofmammalian and yeast Rab proteins is shown in Table II; Rabs shown in bold seem to be

unique to one pathway.[65]

Other Rab proteins have been shown to have a more specialized function or

regulated application, often reflecting differentiated cell function.[66-77] The Rab3

group of proteins have been linked to specialized or regulated exocytotic processes; they

show approximately 80% protein sequence homology, with the differences clustering in

the C-terminal 32 amino acids. This group consists of: Rab3a, involved in synaptic

vesicle and chromatin granule traffic, Rab3b, localized to the polarized epithelial tight

junction, and Rab3c, involved in synaptic vesicle traffic.[78-89] Expression of specific

rab3 proteins has also been implicated in other specialized secretory processes.[90-93]

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Table II

The expression of several isoforms of the small-molecular-weight Rab3 GTP-binding

proteins is a characteristic feature of all cell types undergoing regulated exocytosis, in

which Rab3 proteins are considered to regulate the assembly/disassembly of a fusion

complex between granule and plasma membrane in a positive and negative manner

through interaction with effector proteins. The pattern of Rab3 protein expression may,

therefore, provide a subtle means of regulating exocytosis.[94] Rab2 has been shown tobe important in neuronal adhesion.[89, 95] Rab4 has been suggested to play a role in

insulin-induced GLUT4 translocation.[96] Rab11a and Rab25 associate with the apical

recycling system of epithelial cells; Rab25 may selectively regulate the apical recycling

and/or transcytotic pathways.[97-100] Rab7 levels have been demonstrated to regulate

the rate of antigen presentation in B cells.[101] Rabs 5a and 8a exhibited up-regulation

in addition to Rab3a in oligodendrocytes.[102] Rab32 is a dual function protein that

participates in both mitochondrial anchoring of PKA and mitochondrial dynamics.[103]

Rab dysfunction has been linked to disease - Rab18 may be a proto-oncogene active in

medulloblastoma and Rab27A mutations affecting melanosome transport have been

observed in Griscelli syndrome, [104] [105-108]

Contractile vacuole system and Rab in Dd

The contractile vacuole (CV) is a specialized membrane-bound organelleresponsible for osmoregulation in many fresh water protozoa and amoebae. In Dd

studies have revealed that the CV is a complex of discrete separable bladder-like pump

vacuoles associated with tubular spongiomal network.[4, 109] The vacuoles and the

spongiomal networks (tubules) are associated with the cortical actin network and are

interconvertible.[110, 111] Disruption of Rho, another Ras superfamily member

concerned with actin traffic, causes the development of unusually large CV.[112] Dd

deficient in clathrin heavy-chain lack CV and have impaired osmoregulation.[113]

Capable of moving and fusing, elements of the CV complex collect excess cytoplasmic

water and deposit it to the pump vacuoles. The vacuole periodically fuse to the plasma

membrane with the formation of pores and expel excess water, using a myosin-driven

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contraction that forces water from the vacuole into the extracellular medium without

losing intracellular essential ions.[4, 114-116]

In Dd the CV is enriched in vacuolar H+ATPase, alkaline phosphatase, membrane-

bound calmodulin, and a Ca2+/H+ antiport P-type ATPase;[4, 117-130] some work is

being done, including some inquiries in our laboratories, on characterizing the CV in an

intracellular Ca2+ regulatory capacity.[131-133] The existence of one enzyme to charge

a proton gradient across the CV membrane, another that uses this proton gradient to

modulate a Ca2+ gradient across the same membrane, and the presence of a Ca2+-

sensitive signal transducer all seem highly suggestive of a role for the CV in Ca2+

metabolism in Dd.[132] Another important CV-associated protein, drainin,has beenshown to act as a check point of channel formation between the CV and the plasma

membrane and is required for membrane fusion.[134] An Rh50-like protein and LvsA,

the latter homologous to the mammalian beige/LYST protein, are also found in

association with the CV for as yet uncharacterized reasons, although it has been shown

that LvsA is essential for cytokinesis, endocytosis, and osmoregulation.[135-139] A

chimeric protein termed dajumin-GFP has also been shown to localize to the CV, but the

significance of this has not been determined.[114, 140] Interestingly, dajuminGFP is

the result of the construction of a chimera between a developmental cell-adhesion

molecule, csA, and a transmembrane sequence, whereas DdCAD-1, a 24-kD Ca2+-

dependent developmental cell-adhesion molecule, is transported to the cell surface via

contractile vacuoles.[141] Current evidence seems to indicate that the CV is separate

from the endocytic system in membrane traffic,[27, 114] but there is evidence for cross-

traffic with the acidosome.[142] Studies using vital dyes and fluorescent proteins areoften used to distinguish endosomal and CV compartments in living cells ofDd.[143]

Studies have been performed using the styryl dye FM4-64 and GFP-tagged drainin as

markers of the CV.[114] In our lab we have been using FM1-43 as an effective marker

for the CV.

In Dd at least two members of Rabs, DdRabD and DdRab11a are associated with

the CV networks.[4, 144] RabD, a DdRab14-related GTPase colocalized with vacuolar

H+ATPase in the reticular membranes of the CV complex and in lysosome.[4] Cells

overexpressing RabD dominant negative mutation (RabD N121I) are impaired in their

ability to regulate water homeostasis.[145] Recently we identified anotherDd Rab11

homologue (DdRab11a). Dd Rab11a is exclusively associated with the CV system

undergoing formation, fusion and expulsion of water.[144] Cell lines overexpressing

dominant negative (Dd Rab11N121I) and dominant active (DdRab11Q72L) mutation

revealed functional defects in their osmotic regulation, and displayed morphological

alterations in their CV membrane structure (Mitra et al. unpublished).[144] Together,these observations indicate that both DdRabD and DdRab11a are critical in maintaining

CV morphology and regulating the structure and function of the CV system.

EndocytosisEukaryotic cells appear to uptake material from the environment by two

morphologically distinct processes: phagocytosis and macropinocytosis are

accomplished by development of pseudopodia that engulfs adjacent material, while

receptor-mediated endocytosis and micropinocytosis are accomplished by invagination

of the plasma membrane (PM). Several Rab GTPases have been localized to distinct

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compartments of the endocytic pathway.[146] Rab4 is associated with early endosomes

and recycling vesicles and regulates membrane recycling from early endosomes.[146]

Rab5 is apparently involved in regulating fusion of early endosomes.[147] Rab5a has

been shown to be involved in both endocytosis and phagocytosis, its membrane

association enhanced during increased levels of these processes.[148] Rab5b and Rab5c

colocalize with the both transferrin receptor and Rab5a, stimulate the homotypic fusion

between early endosomes in vitro and increase the rate of endocytosis when

overexpressed in vivo. These data demonstrate that three Rab5 isoforms cooperate in the

regulation of endocytosis in eukaryotic cells.[149] Rab5-GTP is required on both sets of

endosomes for fusion in vitro and in living cells.[150] Rab7 is an intracellular GTPaseinvolved in the regulation of early to late endosome fusion and late endosome to

lysosome membrane transport.[101, 146] It also appears to be involved in maintenance

of the perinuclear lysosome compartment,[151, 152] and is apparently linked to the

cytoplasm via a myosin related protein RILP.[153, 154]

The endocytic pathway inDdis rapid, the maturing endosome undergoing multiple

fusions that leads to post-lysosomes within an hour.[155] Experiments with colloidal

iron has shown 500 endocytic vesicles per cell and supports a maturation process model,

with vesicles displaying different characteristic components (adaptins, acid phosphatase,

vacuolar H+ ATPase, BMP) as maturation progresses.[156] The vacuolar H+ ATPase is

involved in endosomal acidification, with a highly acidic early endosomal compartment

(t1/2 = 18 min; pH 4.3) developing, before progressing into a less acidic late

endosomal/prelysosomal compartment (pH 5.8-6.0). Other constituents of Dd

endosomes include actin, DdRab7, a p34 cysteine protease, and an unidentified 25 kDprotein.[125, 127, 128, 157] Data suggests that endocytic cargo is channeled from

endosomes to secondary lysosomes that are actively linked to the plasma membrane via

recycling vesicles.[158-160] An organelle termed the acidosome has also been described

as responsible for endosome acidification.[142, 161-164] Clathrin heavy-chain deficient

Dd lack endosomes and show impairment of fluid-phase endocytosis, although

phagocytosis remains unaffected.[113] It has been shown that myosin IB functions in

the recycling of plasma membrane components from endosomes back to the cell

surface.[165] There are at least three phosphatidylinositide 3-kinase genes in

Dictyostelium discoideum - DdPIK1, DdPIK2, and DdPIK3; results suggest that

Dictyostelium DdPIK1 and DdPIK2 gene products regulate multiple steps in the

endosomal pathway, and function in the regulation of cell shape and movement perhaps

through changes in actin organization.[166] Caffeine, which discharges Ca2+ stores, has

been shown to inhibit endocytosis, as has La3+, a Ca2+ transport inhibitor.[167] The Dd

cyclin-dependent kinase Crp has been shown to influence rates of fluid uptake andphagocytosis.[168] Bis(monoacylglycerol)phosphate (BMP), a lipid characteristic of

lysosomes in animal cells, comprises up to half of the lipid in the endocytic pathway of

Dd.[169] A study of membrane traffic showed that a steady-state is reached between

PM and endosomes after about 1 hour; 1 PM equivalent of membrane is internalized and

recycled once every 20 minutes during phagocytosis and once every 40 minutes during

pinocytosis.[170]

As might be expected, pinocytosis is highly active in axenic Ddamoebae.[171-173]

The kinetics of pinocytosis inDdwere investigated using FITC-dextran as a fluid-phase

marker. Influx rate was shown to be equivalent to 9 microns3 of fluid/cell x min, with

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saturation of the pinocytotic/exocytotic system within 90 minutes. At equilibrium, the

amount of internalized marker reached a value equivalent to 790 microns3 of fluid taken

up per amoeba, a volume paradoxically higher than the total aqueous space of the cell

(520 microns3), indicating significant concentration of the marker.[174]

PhagocytosisPhagocytosis is an actin-dependent and clathrin-independent process by which cells

internalize particles (>0.3-0.5 m), essential for defense of multicellular organismsagainst invading pathogens and is the major means by which many unicellular organisms

obtain nutrients.[175-177]

Like mammalian macrophages and neutrophils, Ddis a professional phagocyte and

can internalize fluid and particles at a high rate.[24] Studies in Dd have led to the

discovery of many motors involved in critical steps of phagocytosis and membrane

transport. The myosin family of motors, especially the unconventional myosins, interacts

with the actin cortex to facilitate the internalization of external materials during the early

steps of phagocytosis. Members of the kinesin and dynein motor families, which mediate

transport along microtubules (MTs), facilitate the intracellular processing of the

internalized materials and the movement of membrane. Recent studies indicate that some

unconventional myosins are also involved in membrane transport, and that the MT- and

actin-dependent transport systems might interact with each other.[178-180] The class I

myosins contribute to both macropinocytosis and phagocytosis by playing a general role

in controlling actin-dependent manipulations of the actin-rich cortex. A class VII myosin

has been shown to be important for phagocytosis.[175, 181] Phagocytosis happens in anumber of stages, including attachment of particles to the cell surface receptors,

engulfment of particles (dependent on reorganization of plasma membrane and its

cortical cytoskeletal elements), and formation of phagosomes.[182, 183] Experiments

that generated a temporal profile of signaling, cytoskeletal, and trafficking proteins inDd

resulted in: an early phase, characterized by nascent phagosomes associated with coronin

and lysosomal glycoprotein LmpB; at least two phases of delivery of lysosomal

hydrolases (cathepsin D [CatD] and cysteine protease [CPp34]) and removal of plasma

membrane components (PM4C4 and biotinylated surface proteins); and a late maturation

phase characterized by quantitative recycling of hydrolases and association with

vacuolin. This membrane traffic was accompanied by Rab7 and the endosomal SNAREs

Vti1 and VAMP7; also, lysosomal glycoproteins of the Lmp family showed distinct

trafficking kinetics.[184] Vacuolar H+ ATPase gene expression is rapidly enhanced by

phagocytosis, but not by fluid-phase endocytosis.[185] Deletion of the unique Dd Gprotein subunit has been observed to impair phagocytosis but has little effect on fluid-

phase endocytosis; subunit null cells are defective in reshaping the actin network into aphagocytic cup, and eventually a phagosome, in response to particle attachment.

Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited

phagocytosis, suggesting the possible involvement of these effectors in the process.[186]

NEM-sensitive factor (NSF) is an essential protein required during membrane transport;

Dd experiments mutagenizing NSF gene nsfA demonstrated effects on

macropinocytosis, internalization of surface membrane and phagocytosis.[187] At least

six members of the family of Chediak-Higashi/Beige (BEACH) proteins, implicated in

the function of lysosomes, can be identified in Dd. Mutations in LVSA exhibit

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alterations in the organization and function of the early endocytic and phagocytic

pathway, and mutations in LVSB exhibit enhanced regulated secretion of lysosomal

enzymes.[139] AP-1 (adaptor complex-1) is localized to phagocytic cups in in Dd,

recruited to phagosomal membranes at this early stage of phagosome formation and

rapidly dissociated from maturing phagosomes. Mutations disrupted for AP-1 medium

chain display phagocytosis rates decreased by 60%, with impairment of large particle

engulfment predominant; cells exhibiting incomplete engulfment are often observed.

Macropinocytosis is also impaired in apm1(-) cells. .[188]

Dd utilizes macropinocytosis as its major route of fluid uptake from the

environment, with laboratory strains of this organism displaying fluid-uptake rates twoto ten times higher than rates observed in macrophages or neutrophils.[24]

Macropinocytosis refers to the formation of primary large endocytic vesicles of irregular

size and shape, generated by actin-driven evaginations of the plasma membrane,

whereby cells avidly incorporate extracellular fluid. Macropinosomes may fuse with

lysosomes or regurgitate their content back to the extracellular space. In multiple cell

types, macropinocytosis is a transient response to growth factors. When amoebas are

cultured under axenic conditions, macropinocytosis is induced so as to fulfill nutritional

requirements.[189] The process of macropinocytosis is dependent on actin, coronin, andother actin-binding proteins, and has been shown to be regulated by Rac small GTPases

and phosphatidylinositol 3-kinases.[190, 191] Although clathrin does not appear to be

associated with phagocytosis, disruption of the clathrin heavy chain leads to an 80%

reduction in fluid uptake.[192, 193] Studies using consecutive labeling with two

fluorescent fluid-phase markers demonstrated that within the first few minutes newmacropinosomes underwent fusion with pre-existing endosomes. The fusing endosomes,

which represent the mixing compartment, displayed extreme shape changes and rapid

transport about the cell in association with microtubules. Treatment of cells with agents

that selectively disrupted either actin filaments or microtubules confirmed that endosome

dynamics were microtubule based. Further maturation of endosomes led to loss of

pleiomorphy in favor of a spherical shape, inability to fuse with new macropinosomes,

and diminished motility.[194]

Recent studies have identified a large number of Ras, Rho and Rab related small

GTPases inDdassociated with the phagocytic event. Among the Rabs, RabD, Rab7 and

Rab11 strongly involve in regulating phagocytosis. [144, 191, 195] RabD, aDdRab14-

like GTPase are enriched in phagosome, enhance phagosomal fusion and maturation. In

cells overexpressing the dominant-active mutation Q67L, the rate of phagocytosis is

increased by 2-fold and the rate of phagosome-phagosome fusion is five times higher as

compared to wild-type cells. Whereas the dominant-negative mutation N121I,phagocytosis and phagosomal fusion is reduced almost by 50%. In addition, the

dominant active mutation causes multiparticle phagosome formation, suggesting RabD

plays a significant role in regulating homotypic phagosome fusion in

Dictyostelium.[195] DdRab7 has been shown to localize to the early phagosome, and

plays an important role in regulating early and late steps of phagosomal

maturation.[191] Rate of phagocytosis as well as lysosomal enzymes such as LmpA

(lysosomal integral membrane protein) and a-mannosidase enzyme is lower in the cell

line overexpressing dominant negative mutation Rab7T22N.[191, 196] Dd mutants

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lacking RasS are impaired in phagocytosis, fluid-phase endocytosis, and growth, but

show an enhanced rate of cell migration.[197]

InDdat least two Rab proteins, DdRab7 and DdRabD have been shown to regulate

macropinocytosis in a positive fashion. DdRab7 is directly implicated in late endosomal

events, and DdRabD is most directly associated with contractile vacuole function, but

disruption of these downstream effectors may still affect macropinocytosis rates.[157,

196]

Exocytosis from post-lysosomesAfter completion of early and late steps of endosomal pathway and digestion of

nutrients, the fluid phase enters into big, nearly neutral post-lysosomal vesicles where

indigestible particles, soluble components, lysosomal enzymes like -mannosidase and

-glucosidase are concentrated and returned to the cell surface.[157, 198, 199] Therelease of both particles and fluid happen within seconds.[23, 125, 198, 200] Later

empty vesicle remains at the site of exocytosis and it is believed that vesicular membrane

collaspe when exocytosis is complete.[23, 201] Lysosome and post-lysosome is

surrounded by actin cytoskeleton, subunits of V-H+ATPase and some members of Rab

GTPases. The post lysosomal compartment is characterized by two isoforms of

vacuolin, A and B, which are encoded by different genes and associate with the

cytoplasmic side of post-lysosomal vacuoles and patches on the plasma membrane in

vivo. In mutants of Vacuolin B endocytosis is normal, but the progression of fluid-phase

marker from acidic to neutral pH is impaired. Furthermore, in the mutants post-

lysosomal vacuoles are dramatically increased in size and accumulate endocytic marker,suggesting a role for vacuolin B in targeting the vacuole for exocytosis.[202] In other

work, GFP-vacuolin-decorated vesicles were identified as a post-lysosomal compartment

that acquires endocytic markers shortly before exocytosis. At earlier stages, this post-

lysosomal compartment was identified by the binding of a tagged cytoskeletal protein,

coronin-GFP. Vacuoles were coated with filamentous actin along the entire post-

lysosomal pathway, and the integrity of the actin coat was required for exocytosis.[203]

InDdtwo Rabs, RabD and Rab7 are associated with the exocytosis process. Rab7 is

enriched in lysosome and post-lysosome fractions and regulates membrane flow at the

late steps of endosomal pathway, possibly between lysosomes and post-lysosomes.[196]

Cell line overexpressing dominant negative mutation of Rab7T22N causes slower rate of

exocytosis of the fluid phase and oversecret the lysosomal enzyme, -mannosidase,whereas in dominant active mutation of Rab7Q67L exocytose fluid phase faster than

control cell.[196] This suggests that DdRab7 regulate retrograde transport of lysosomalenzymes and the vacuolar H+ATPase from post lysosomes to lysosomes coupled with

the efficient release of fluid phase from cells. RabD is localizes to the endosomal

pathway and the CV membrane system inDd. However, the dominant negative mutation

of RabDN121I significantly slower the rate of exocytosis.[145]

Endoplasmic reticulum and golgi pathwaysEvidence suggests that a single round of vesicular transport between the ER and the

Golgi involves a rapid transit through NSF and GTP sensitive step(s) involved in vesicle

formation or transport to a novel intermediate compartment, followed by a regulated

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fusion event triggered in the presence of Ca2+ and functional components interacting

with member(s) of the Rab gene family.[204] Numerous trafficking proteins have been

identified in this series of pathways, including KDEL receptors, p24 family members,

SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two

SCAMPs.[37, 205, 206] Golvesin is a new protein associated with membranes of the

Golgi apparatus and post-Golgi vesicles in Ddcells.[207] In Saccharomyces cerevisiae,

clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a

process that involves cycling of TGN proteins between the TGN and endosomes.[208]

Rab1 and Rab2 are associated with ER to Golgi transport, whereas Rab6 has been

shown to be distributed along the exocytic pathway in association with the medial andtrans regions of the Golgi apparatus.[63, 209-211] Traffic also occurs between the Golgi

and endosomes. Endosome to Golgi traffic seems to be regulated by Rab9 and

Rab11.[212, 213]

Multicellular developmentDd amoebae grow as separate, independent cells but interact to form multicellular

structures when challenged by adverse conditions such as starvation. Up to 100,000

cells signal each other by releasing the chemo-attractant cAMP and aggregate together

by chemotaxis to form a mound that is surrounded by an extracellular matrix. This

mechanism for generating a multicellular organism differs radically from the early steps

of metazoan embryogenesis. However, subsequent processes depend on cell-cell

communication in both Dd and metazoans. Many of the underlying molecular and

cellular processes appear to have arisen in primitive precursor cells and to have remainedfundamentally unchanged throughout evolution. Basic processes of development such

as differential cell sorting, pattern formation, stimulus-induced gene expression, and

cell-type regulation are common to Dd and metazoans. The entire developmental

program of Dd occurs in approximately 24-hours, accelerating experimental time

courses.

A form of sexual phagocytosis, where zygote giant cells develop and serve as foci

for further development by chemoattracting and cannibalizing hundreds of local

amoebae, occurs in Dd; this sexual phagocytosis bears similarities to and differences

from asexual endocytosis. Data indicates that a glucose-type receptor is involved in

selective uptake ofDdamoebae by giant cells.[214] During development, a second form

of a number of lysosomal enzymes begins to accumulate. The second ( 'late') form of

these enzymes differs from the pre-existing ('early') form in post-translational

modification. Pulse-chase experiments using [35S]methionine show that the late form ofalpha-mannosidase-1 is made by synthesis de novo starting 8 hours after the onset of

development. Experiments show there is no interconversion between early and late

forms in vivo, and the two forms have a similar half-life in vivo when measured during

the same period of development. It is suggested that the late form lacks the mannose 6-

phosphate residue required for efficient uptake.[215] Clathrin-minus cells have been

shown to be delayed in early development; when exposed to starvation conditions, such

cells stream and aggregate more slowly than wild-type cells. Although clathrin-minus

cells display only 40% the level of extracellular cyclic AMP binding normally found in

wild-type cells, they respond chemotactically to extracellular cyclic AMP. Clathrin-

minus cells down-regulate cyclic AMP receptors, but only to half the extent of wild-type

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cells. It was found that the extent of development of clathrin-minus cells was variable

and influenced by environmental conditions, with the final structure varying from a

finger-like projection to a short, irregular fruiting body. Microscopic examination of

these terminal structures revealed the presence of intact stalks but a complete absence of

spores. Clathrin-minus cells expressed prestalk (ecmA and ecmB) and prespore (psA and

cotB) genes normally, but were blocked in expression of the sporulation gene spiA.

Only partial sorting of clathrin-minus prestalk and prespore cells was observed to occur.

Even when mixed with wild-type cells, clathrin-minus cells failed to sort correctly and

never constructed functional spores.[216] The DdRtoA protein links both initial cell-

type choice and physiological state to cell-cycle phase; rtoA- cells generally do notdevelop past the mound stage, and have an abnormal ratio of prestalk and prespore cells.

RtoA is also involved in fusion of endocytic/exocytic vesicles. Cells lacking RtoA,

although having a normal endocytosis rate, have a decreased exocytosis rate and

endosomes with abnormally low pHs. RtoA levels vary during the cell cycle, causing a

cell-cycle-dependent modulation of parameters such as cytosolic pH.[217]

The expression of rabE inDdhas been found to be under developmental regulation,

with an onset of message expression after 8 h of development. Comparison of the rabE

amino acid sequence with the database showed that its unique domains were most

similar to the products of four mammalian rab genes. Interestingly, only the rabE protein

and its four mammalian homologs contained the sequence WDIAGQE, a variation of the

conserved Rab P/M 3 domain WDTAGQ.[218] Mutant strains ofDdthat fail to develop

spores in a pure population but sporulate well in chimerae with wild type cells have

revealed a gene with a region of high similarity to the Rab family of small GTPases and1 transmembrane domain, thought to have a cell-autonomous role in prestalk A cell

differentiation.[219]

Protein interactionsSome of the proteins known to interact with Rabs fall into the following categories:

I. Active state modifiersA. GAP GTPase activating proteins

GTPase activating proteins, or GAPs, accelerate the low intrinsic GTP hydrolysis

rate of typical Ras superfamily members, thus causing their inactivation. Typically

associated with the target membrane compartment. A mechanism has been proposed in

which GAPs provide a catalytic arginine ('arginine finger') in trans to accelerate the GTP

hydrolysis rate of the transport GTPases.[220] Work with p21 Ras has shown thatinteractions with GAP occur in L2-2 (coincident with P/M2 and SW1) and L4 (overlapwith P/M3 and SW2).[221-224] In yeast, there is a large family of Ypt/Rab-GAPs,

members of which discriminate poorly between GTPases involved in regulating different

steps of exo- and endocytic transport routes.[225] Mutations in GAPs have been

implicated in two common genetic disorders associated with an increased cancer risk,

neurofibromatosis-1 and tuberous sclerosis.[226]

Dd RacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it

shares with mammalian Bcr and Abr. Experimental results have indicated that DRG

modulates F-actin dynamics and cAMP-induced F-actin formation via Rac1-dependent

signalling pathways. DRG's RacE-GAP activity is required for proper cytokinesis to

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occur. It is additionally indicated that the specificity of DRG is not limited to members

of the Rho family of small GTPases. A recombinant DRG-GAP accelerates the GTP

hydrolysis of RabD 30-fold in vitro, and complementation studies show that DRG-GAP

activity is required for the RabD-dependent regulation of the CV.[227, 228]

B. GEF Guanine nucleotide exchange factors

Also known as guanine nucleotide dissociation stimulators (GDS), these proteins

activate Ras superfamily members by accelerating GTP for GDP exchange.[229] An

essential component of the source membrane compartment. The mammalian Mss4

protein, a typical GEP, stimulates GDP release from both yeast and mammalian Rabs,specifically Rab1, Rab3, Rab8, Rab10, Sec4 and Ypt1 but not Rab2, Rab4, Rab5, Rab6,

Rab9 and Rab11. A mutation in the zinc-binding domain of Mss4 (Mss4 D96H), a

region that is highly conserved between Mss4 and its yeast homologue Dss4, completely

abolished its property to bind to, and promote GDP-GTP exchange in Rab3a.[230]The gene that encodes DdRasGEFB, a protein with homology to known RasGEFs

such as the Son-of-sevenless (Sos) protein has been reported. Cells in which the gene

for RasGEFB was disrupted moved unusually rapidly, but lost the ability to perform

macropinocytosis and therefore to grow in liquid medium. Crowns, the sites of

macropinocytosis, were replaced by polarised lamellipodia. Mutant cells were also

profoundly defective in early development, although they eventually formed tiny but

normally proportioned fruiting bodies. This defect correlated with loss of discoidin

Igamma mRNA, a starvation-induced gene, although other genes required for

development were expressed normally or even precociously. RasGEFB was able torescue a Saccharomyces CDC25 mutant, indicating that it is a genuine GEF for Ras

proteins.[231]

C. GDI Guanine nucleotide dissociation inhibitors

Rab GDP dissociation inhibitors (GDIs) play the reverse role of GEFs they slow

the exchange of GTP for GDP and thus tend to hold Ras superfamily members in an

inactive state. These proteins are also implicated in regulating the association between

small GTPases and membranes.[232-234] The small family of Rab-GDI identified

consists of several closely related isoforms, the functional differences between which are

still largely unknown.[235] There is significant sequence homology between GDIs and

REPs, and they seem to compete for an overlapping binding site on Rabs, possibly

involving the switch domains, L3-3, and the C-terminal hypervariable region.[236]Although REP and GDI share common Rab-binding properties, GDI cannot assist in Rab

prenylation and REP cannot retrieve Rab proteins from the membranes.[237] Disease

states such as non-specific X-linked mental retardation (MRX) is a very common

disorder which affects approximately 1 in 600 males, have been linked to GDI

mutations.[238]

Two Rho GDI homologues inDdhave been identified, with GDI1 sharing 51-58%

sequence homology to RhoGDIs from diverse species and GDI2 sharing 40-44%

sequence homology. Besides being more divergent, GDI2 lacks the N-terminal

regulatory arm characteristic for RhoGDI proteins. Both are cytosolic proteins and do

not relocalize upon reorganization of the actin cytoskeleton. Rac1a/1b/1c, RacB, RacC

and RacE were identified as interacting partners for GDI1. Cells lacking GDI1 are

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multinucleate, grow slowly and display a moderate pinocytosis defect, but rates of

phagocytosis are unaffected. Mutant cells present prominent actin-rich protrusions, and

large vacuoles that are continuous with the contractile vacuole system. The actin

polymerization response upon stimulation with cAMP was reduced, but the motile

behavior toward the chemoattractant was unaffected.[112]

II. Membrane targetting and insertionA. REPs Rab escort proteins

Rab escort proteins (REPs) 1 and 2 are closely related mammalian proteins required

for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric RGGTII complex.[239-241]

REPs share three N-terminal structurally conserved regions with GDIs; three C-

terminal conserved regions are uniquely characteristic of REPs.[242] Data shows that

REP alone is sufficient to chaparone prenylated Rabs to membrane insertion points; REP

also shows a 20-times higher affinity for the GDP-bound form over the GTP-bound

form.[243] In contrast, GDIs chaperone cytosolic Rabs cycling through the inactive

state, and there is a competition between the two for binding to some Rabs.[244]

Choroideremia is an X-chromosome-linked disease that leads to the degeneration of the

choriocapillaris, the retinal pigment epithelium and the photoreceptor layer in the eye.

The gene product defective in choroideremia, CHM, is identical to Rab escort protein 1

(REP1).[245-249]

B. RGGT- Rab geranylgeranyl transferase IIMammalian cells seem to possess at least three protein prenyltransferases (CAAX

farnesyltransferase, CAAX GG transferase, and RGGT) that are specific for different

classes of low molecular weight GTP-binding proteins and other proteins.[47] RGGT, a

heterodimer, attaches 20-carbon geranylgeranyl groups to cysteine residues in Rab

proteins that contain the C-terminal sequence XXXCC, XXCCX, XCCXX, CCXXX,

XXCXC, or occasionally CXXX;[48, 240, 250] L3/3 and the C-terminal hypervariableregion, but not the switch domains, are required for efficient processing by

RabGGTase.[236] According to the currently accepted model for their action, newly

synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to

RGGT which covalently modifies the Rab protein with two geranylgeranyl moieties.

Geranylgeranylation of these proteins on C-terminal cysteine motifs is crucial for their

membrane association and function.[251-253] After prenylation, the Rab protein remains

in complex with REP and is delivered to the target membrane by the latter.[254] Notsurprising considering its interaction with REP, RGGT also binds to GDI, which acts as

a chaparone for Rab during the cytoplasmic transit from target membrane compartment

to source membrane compartment.[255]

C. PRA Prenylated Rab acceptors

Prenylated Rab acceptors (PRA) are proteins that bind prenylated Rab GTPases and

inhibit their removal from the membrane by GDI. The two PRA isoforms known show

distinct intracellular localization with PRA1 localized primarily to the Golgi complex

and PRA2 to the endoplasmic reticulum (ER) compartment.[256, 257]

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III. Cytoskeletal componentsRho and Rac small GTPases have been implicated in the control of actin trafficking

and polymerization, and as such may frequently interact with Rab proteins in vesicle

trafficking processes.[258-267] In yeast, mutational analysis of the Rho3 effectordomain reveals three distinct functions in cell polarity: regulation of actin polarity,

transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of

vesicles with the plasma membrane, and evidence has been found that the vesicle

delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the

docking and fusion function is mediated by the exocyst component Exo70, with Rho

GTPase Cdc42 acting as an allosteric regulator of vesicle docking and fusion apparatusto provide maximal function at sites of polarized growth.[268, 269] Rab4, which seems

to be involved in retrograde early endosome to plasma membrane traffic, has been

shown to interact with cytoplasmic dynein, a retrograde motor protein.[270] The class I

myosins contribute to both macropinocytosis and phagocytosis by playing a general role

in controlling actin-dependent manipulations of the actin-rich cortex. A class VII myosin

has been shown to be important for phagocytosis.[175, 181] Dd MyoM has recently

been tied to the signaling pathways controlling actin cytoskeleton remodeling.[271]

Other results have shown that three myosin I isoforms - myoB, myoC, and myoD - share

in supporting endocytosis.[272, 273] The microtubule associated dynamins form a

family of multidomain GTPases involved in endocytosis, vesicle trafficking and

maintenance of mitochondrial morphology.[274] The identification and functional

characterization ofDddynamin A (dymA), a protein composed of 853 amino acids that

shares up to 44% sequence identity with other dynamin-related proteins, has beendescribed. Dynamin A is present during all stages ofDd development and is found

predominantly in the cytosolic fraction and in association with endosomal and

postlysosomal vacuoles. Cells lacking a functional copy of dymA show alterations of

mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake.

They also become multinucleated due to a failure to complete normal cytokinesis. These

pleiotropic effects of dynamin A depletion can be rescued by complementation with the

cloned gene. Morphological studies using cells producing green fluorescent protein-

dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles.

Double labeling with vacuolin, a marker of a postlysosomal compartment in D.

discoideum, showed an almost complete colocalization of vacuolin and dynamin A.[275]

A kinesin-like protein that interacts with Rab6-GTP has been identified. This protein,

termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in

the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which

contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular

transport. Thus, a molecular motor is a potential effector of a Rab protein, and

coordinated action between members of these two families of proteins could control

membrane dynamics and directional vesicular traffic.[276-278] Disruption of Scar, a

member of the WASp protein family, has been shown to reduce the levels of cellular F-

actin by 50%, significantly reduce rates of fluid phase macropinocytosis and

phagocytosis, delay exocytosis of fluid phase, and delay movement of fluid phase from

lysosomes to post-lysosomes; fluorescence microscopy reveals that some endo-

lysosomes are ringed with F-actin in control cells but no F-actin can be detected

associated with endo-lysosomes in Scar null cells. Disruption of the scar gene in a

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profilin null background results in greater decreases in the rate of fluid phase

internalization and fluid phase release compared to either mutant alone, implying that

Scar and profilin functionally interact to regulate internalization of fluid and particles

and later steps in the endosomal pathway, probably through regulation of actin

cytoskeleton polymerization.[279] Dd mutants lacking coronin are impaired in

cytokinesis and all actin-mediated processes. Analysis of coronin-GFP (green-

fluorescent protein) fusions and knockout mutants shows that coronin participates in the

remodelling of the cortical actin cytoskeleton that is responsible for phagocytosis and

macropinocytosis.[179]

IV. Vesicle targetting, tethering and fusionA. SNAPs - Soluble NSF attachment proteins

The soluble N-ethylmaleimide-sensitive-factor-attachment proteins (SNAPs) are

eukaryotic soluble proteins required for membrane fusion. Based on their initial

identification in bovine brain cytosol, they are divided in alpha/beta and gamma

subfamilies. SNAPs act as adapters between N-ethylmaleimide-sensitive factor (NSF), a

hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the

NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP-driven

conformational change in the SNAREs, resulting in dissociation of the complex. Results

in yeast have identified Sec9 as the yeast cognate of SNAP-25 and suggest that SNARE

complexes acting at specific stages of vesicular transport serve as the ultimate targets of

regulation by members of the Sec4/Ypt1/Rab family of GTPases.[280]

Immunoprecipitation using c-myc-tagged NSF has revealed two associatedpolypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) identified

as the Dd homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-

SNAP molar ratio is close to 3 in vegetative amoebae from this organism.[281]

B. SNAREs - Soluble NSF attachment protein receptorsMembrane fusion requires the formation of a complex between a vesicle protein (v-

SNARE) and the target membrane proteins (t-SNAREs). Syntaxin 4 is a t-SNARE that,

according to previous overexpression studies, is predominantly localized at the plasma

membrane; studies have also shown endogenous syntaxin 4 found in intracellular

vesicular structures in addition to regions of the plasma membrane. In these vesicular

structures syntaxin 4 colocalized with rab11, a marker of recycling endosomes.

Furthermore, syntaxin 4 colocalized with actin at the dynamic regions of the plasma

membrane. Treatment with N-ethylmaleimide, the membrane transport inhibitor, caused

an increased accumulation of syntaxin 4/rab11 positive vesicles in actin filament-like

structures. Finally, purified recombinant syntaxin 4 but not syntaxin 2 or 3 cosedimented

with actin filaments in vitro, suggesting direct interaction between these two

proteins.[282] In homotypic TGN fusion, the Tlg SNARE complex composed of Tlg1p,

Tlg2p, Vti1p, and the rab Vps21p was shown to be implicated.[283] In yeast, the

ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are

distributed similarly but localize primarily with Golgi membranes. All of these SNARE

proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of

SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently

packaged into vesicles under these conditions. Vesicles bearing mutations in Bet1p or

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Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes

containing these mutations are fully functional. In contrast, vesicles bearing mutations in

Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these

mutations block fusion. When a transmembrane-anchored form of Ypt1p is used to

restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain

competent for fusion.[284]

ADdsyntaxin 7 homologue (26% identity and 54% similarity to human syntaxin 7)

has been identified and shown to be able to form a complex with NSF and alpha- and

gamma-SNAPs. Most of D. discoideum syntaxin 7 seems to be associated with

endosomes and was shown to be necessary for homotypic fusion.[285] The syntaxin 7complex contains two co-t-SNAREs [Vti1 (Vps10p tail interactor 1) and syntaxin 8] and

a v-SNARE [VAMP7 (vesicle-associated membrane protein 7)]. In endosomes and in

vitro, syntaxin 7, Vti1 and syntaxin 8 form a complex that is able to bind VAMP7. The

lysosomal content of syntaxin 7, Vti1, syntaxin 8 and VAMP7 is low compared with that

in endosomes, implying a highly active recycling or retention mechanism. It is

suggested that VAMP7 is a v-SNARE present on vesicles carrying lysosomal enzymes,

and that the syntaxin 7-Vti1-syntaxin 8 t-SNARE complex is associated with incoming

endocytic material.[286]

C. Tethering proteinsGolgins are a family of coiled-coil proteins associated with the Golgi apparatus

necessary for tethering events in membrane fusion and as structural supports for Golgi

cisternae. Recent work has shown that golgins such as GM130, golgin-45 and p115 bindto Rab GTPases via their coiled-coil domains, and that GM130, rather than being part of

a static structural matrix, is in dynamic exchange between the membrane surface and the

cytoplasm. Golgins such as bicaudal-D1 and -D2 bind to Rab6, but, rather than tethering

membranes together, link vesicles to the cytoskeleton, thus adding a new function for

this class of proteins. Other golgins containing the Golgi targeting GRIP domain, rather

than binding Rabs, interact with and are recruited to membranes by another class of

GTPase, the Arls. Current evidence therefore suggests that golgins function in a variety

of membrane-membrane and membrane-cytoskeleton tethering events at the Golgi

apparatus, and that all these are regulated by small GTPases of the Rab and Arl

families.[287]

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