FEBS Letters 583 (2009) 3738–3745

journal homepage: www.FEBSLetters.org

Review

Evolution and diversity of the Golgi body

Kevin Mowbrey, Joel B. Dacks *
Department of Cell Biology, University of Alberta, Edmonton, Canada T6G 2H7

a r t i c l e i n f o a b s t r a c t

Article history: Often considered a defining eukaryotic feature, the Golgi body is one of the most recognizable and
Received 20 August 2009 functionally integrated cellular organelles. It is therefore surprising that some unicellular eukary-
Accepted 11 October 2009 otes do not, at first glance, appear to possess Golgi stacks. Here we review the molecular evolution-
Available online 20 October 2009
ary, genomic and cell biological evidence for Golgi bodies in these organisms, with the organelle
likely present in some form in all cases. This, along with the overwhelming prevalence of stacked
Edited by Daniela Corda
cisternae in most eukaryotes, implies that the ancestral eukaryote possessed a stacked Golgi body,
with at least eight independent instances of Golgi unstacking in our cellular history.
Keywords:
Protist
Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Phylogeny
Parasite
Archezoa

1. Introduction Stephanopogon are found free-living in freshwater or marine envi-

The first description by Camillo Golgi of the ‘internal reticular
apparatus’ that would later bear his name [1] was a seminal point
in the field of cell biology. The Golgi body is the central organizing
nexus of the membrane-trafficking system: the crossroads of the
endocytic and exocytic pathways. It is the site of protein glycosyla-
tion and their sorting into vesicles, targeting them to their eventual
cellular destinations. It is a signaling scaffold, a site of biosynthesis,
and many other functions that have been described in exquisite de-
tail in the 110 years since the organelle’s original description [2]. Its
characteristic morphology, with stacks of discoid, flattened, mem-
branous compartments, ‘‘reminiscent of a stack of pita bread” [2]
is one of the most recognizable features of the eukaryotic cell.
The presence of an organelle bearing this morphology is often con-
sidered a fundamental characteristic of eukaryotes.
So it is probably surprising to many that there are some unicel-
lular eukaryotes that lack visible Golgi stacks [3–5]. These ‘Golgi-
lacking’ protists include some relatively well-known parasites such
as Giardia intestinalis, Entamoeba histolytica, microsporidia, heterol-
obosea and various apicomplexans such as Theileria, Cryptosporidi-
um and Babesia (Fig. 1). On the other hand, some of the organisms
and lineages are more obscure, but no less interesting [5]. The oxy-
monads and retortamonads are single-celled commensals, or para-
sites of veterinary importance. Mastigamoeba, Leukarachnion and
Abbreviations: CWP, cyst wall protein; DHFR–TS, Dihydrofolate reductase and
Thymidilate synthase; ESV, encystation-specific vesicle; GRASP, Golgi reassembly
stacking protein; SAR, Stramenopile, Alveolate and Rhizaria
* Corresponding author. Address: 6-30 MSB, Department of Cell Biology, Univer-
sity of Alberta, Edmonton, Canada T6G 2H7. Fax: +1 780 492 0450.
E-mail address: joel.dacks@ualberta.ca (J.B. Dacks).
ronments (Fig. 1). Each cell is at the same time beautiful and puz-
zling, and raises important questions about the diversity and
evolution of the Golgi complex. Do Golgi bodies exist in these
organisms of a morphology that is not immediately recognizable?
If not, then is the lack of the organelle due to primitive absence,
that is, there has never been a Golgi body in the organism or its
ancestors, or is it due to secondary loss, i.e. the Golgi body was
there at one time but has been lost? Even if Golgi bodies do exist
in these cells, then similar questions about the primitive or derived
status of Golgi morphology still remain. What do these organisms
tell us about the Golgi body in the Last Common Eukaryotic Ances-
tor and about the history of the organelle since that ancestor
approximately 1–1.5 billion years ago [6]?
It is important to point out that, by giving these organisms the
descriptor of ‘Golgi-lacking’ here, we are not positively saying that
they are without the organelle. Rather, they all lack visible Golgi
stacks and many were at one time proposed as lacking the orga-
nelle. In this review we will describe these wonderful creatures
and the three lines of evidence for their possession of cryptic Golgi
organelles; phylogenetic, genomic and cell biological. From this,
we will deduce what we can about the ancestral state of the Golgi
body and look to new directions of research in the evolution of
these fascinating organelles.
2. ‘Golgi-lacking’ eukaryotes are spread throughout the
eukaryotic tree and embedded in clades of stacked Golgi-
possessing organisms
Logically, there are two evolutionary possibilities for each of
these candidate lineages. On the one hand, ‘Golgi-lacking’ lineage

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2009.10.025
 K. Mowbrey, J.B. Dacks / FEBS Letters 583 (2009) 3738–3745 3739

Fig. 1. Rogues gallery of ‘Golgi-lacking’ eukaryotes. Micrographs of various ‘Golgi-lacking’ organisms discussed in this manuscript. (A) The heterolobosean Naegleria gruberi
image courtesy of L. Fritz-Laylin, (B) the diplomonad Giardia intestinalis, image courtesy of S.C. Dawson and S. House, (C) the pelobiont Mastigamoeba balamuthi (unpublished
image courtesy of G. Walker), (D) the heterolobosean Stephanopogon minuta image taken from http://www.tolweb.org/Stephanopogon_minuta/129961 with permission from
B. Leander, (E) the entamoebid Entamoeba moshkovskii (unpublished image courtesy of C.G. Clark and (F) the oxymonad Monocercomonoides sp., unpublished image courtesy
of V. Hampl.

could represent a primitive or ancestral state. On the otherhand, if
a ‘Golgi-lacking’ lineage is embedded within a group of organisms,
all with stacked Golgi body, then the simplest explanation is that
Golgi bodies were either lost or modified to an unstacked morphol-
ogy in that candidate ‘Golgi-lacking’ line.
The former concept was formalized as part of the Archezoa
hypothesis [3,7]. This paradigmatic idea proposed that, since
eukaryotes likely evolved from prokaryotic-like ancestors, and
since there were distinguishing eukaryotic-specific cell biological
differences such as Golgi bodies, peroxisomes and principally mito-
chondria, that there might have existed eukaryotes that evolved
away from the main eukaryotic line before the invention of certain
organelles. If this were the case then these basal lineages may have
left behind descendents (Archezoa) that are cytologically simpler
than many eukaryotic cells today, lacking particular organelles, be-
cause they never had them! This was the interpretation for many
of the ‘Golgi-lacking’ organisms, particularly microsporidia, Ent-
amoeba, oxymonads and diplomonads [3]. One prediction was that
these organisms should be the deepest branches in phylogenetic
trees, a prediction borne out when the first molecular phylogenetic
analyses of rDNA and protein coding genes were published [8,9].
However, despite its logical consistency with the data available
at the time and its tremendous contribution to pushing forward
our understanding of evolutionary cell biology, new evidence
now strongly refutes the Archezoa hypothesis. This evidence came
in two types. The first was the discovery of mitochondrial homo-
logues in many of the ‘‘simpler” archezoan taxa (as reviewed in de-
tail elsewhere [10]). The second was the increased sampling of
molecular sequence data and the application of biologically rele-
vant models of sequence evolution to its analysis. For reasons of
computational tractability, the early analyses of molecular se-
quence made some very basic assumptions: all positions in a gene
evolved at similar rates, the same gene between different taxa
evolved at similar rates and all changes between states were
equally likely. When more sophisticated models of sequence evo-
lution were developed, studies showed that that the basal position
the Archezoa was due to rapidly evolving gene sequences that the
programs misinterpreted as relatedness with prokaryotic out-
groups (e.g. [11]).
The Archezoa hypothesis used both ultrastructural and se-
quence data but eventually fell due to the incorporation of incor-
rect assumptions well as the limitations of molecular sampling at
the time. New taxonomic studies now use the best available ultra-
structural information (similarities in cytoskeletal organization,
organelle structure) as well as vastly increased gene sequence
datasets, made possible by genome sequencing. These multi-gene
concatenated phylogenies involve the analysis of 10s to 100s of
genes, often strung end to end and treated as a single polypeptide
sequence (e.g. [12–14]). This has now allowed the relatively stable
establishment of six major eukaryotic supergroups: Opisthokonta,
Amoebozoa, Excavata, Chromalveolata, Rhizaria and Archaeplast-
ida [15]. Furthermore, significant progress has been made recently
in producing a resolved set of relationships between and within
these supergroups. The opisthokonts and Amoebozoa are united
in a larger assembly called the unikonts [16] which appears quite
robust to the application of new data, based on multi-gene concat-
enated phylogenies [12,13]. The stramenopiles and alveolates that
formed the core of the chromalveolates are, in fact, robustly related
to the Rhizaria in several concatenated gene analyses forming the
Stramenopile, Alveolate and Rhizaria (SAR) clade, and these are
strongly resolved as related to the Archaeplastida and the crypto-
phytes and haptophytes in a ‘‘megagroup” [12,17]. And so set
against this framework of eukaryotic relationships, we can now
examine the placement for the various proposed ‘Golgi-lacking’
organisms.
2.1. Microsporidia
Microsporidia are obligate intracellular parasites known to in-
fect a spectrum of animals. Often considered an opportunistic
3740 K. Mowbrey, J.B. Dacks / FEBS Letters 583 (2009) 3738–3745
pathogen, the microsporidian Encephalitozoon cuniculi has been
implicated in causing digestive and nervous system-related syn-
dromes in immunocompromised patients. Apart from their bio-
medical impact on humans, microsporidia also pose threats to
populations of fish and honeybees, the decline of which already
have far-reaching ecological and economic ramifications. Contain-
ing a simple endoplasmic reticulum, a nucleus, and a modified
mitochondrial homologue (mitosome), the microsporidian cell is
dominated by the polar tube which, along with the polarplast
and the posterior vacuole, is used by the parasite to infect host cells
[18]. Once thought to be a prime candidate as the earliest eukary-
otic lineage [9], the microsporidia were amongst the first of the
archezoan lineages to have their evolutionary affinity pinpointed
as more gene sequences were analyzed and the corrections for ra-
pid sequence evolution incorporated into phylogenetic analyses
[19]. It is now clear that the microsporidia are highly divergent
fungi, likely relatives of the zygomycetes [19]. Despite the well-
known unstacked nature of Golgi cisternae in the model organism
Saccharomyces cerevisiae, most fungi do, in fact, possess rather nor-
mal looking Golgi stacks (Fig. 3) [20]. Additionally, fungi are mem-
bers of the supergroup Opisthokonta and sister taxa to the Metazoa
(Fig. 2), which possess either many Golgi stacks in basal lineages or
single Golgi ribbons in higher Metazoa.
2.2. Entamoebae and pelobionts
As the causative agent of amoebiasis, the protozoan parasite
Entamoeba histolytica is of great medical importance, particularly
in the developing world. E. histolytica’s adaptations of a mitosome
and reduction or elimination of mitochondrial metabolic path-
ways, as well as the use of oxidative stress enzymes, enable E. his-
tolytica to prosper in anaerobic environments of the colon of the
host [10]. Found in anaerobic environments of quite a different sort
(mostly anoxic mud and sand, though one lives in the rectum of
frogs), the pelobionts are a group of free-living amoebae, most eas-
ily recognized in their flagellated trophic stage. Most have an
Babesia bovis
Plasmodium
falciparum Toxoplasma
gondii Mastigamoeba
dinoflagellates Cryptosporidium balamuthi
parvum
Tetrahymena
Phytophthora thermophila
ramorum
Rhizaria
Leukarachnion sp.
red algae
land
plants Naegleria
gruberi
haptophytes
cryptophytes
green algae Trypanosoma
brucei
hypermastigotes
Trichomonas
vaginalis
retortamonads
Fig. 2. Eukaryotic phylogeny with emphasis on our Rogues gallery. This depiction of eukaryotic phylogeny shows the relationships of the taxa described in this review. In
cases where a group is not discussed in detail (e.g. land plants), the name of the larger taxonomic group is simply given to reduce complexity of the figure. The red X marks the
spot of a deduced loss of cisternal stacking in the Golgi bodies. This is the most parsimonious explanation for the data and in cases, such as the diplomonads and
retortamonads, two independent instances of unstacking could be possible. Nonetheless, even if the root of eukaryotes were to lie on a ‘Golgi-lacking’ lineage, there has been a
minimum of eight independent instances of Golgi unstacking in the history of eukaryotes.
amoeboid cell body and a single, long flagellum; but one, Pelomyxa,
is a giant multinucleate amoeba covered in tiny non-functional fla-
gella [21]. These organisms are also cytologically simplified with
no visible Golgi bodies and only recently identified mitochondrial
homologues [22]. Each group has been proposed as ancient eukary-
otes at different times; they are indeed sister taxa [23], but embed-
ded within the larger amoebozoan supergroup [15]. This
assemblage includes lobose amoebae, organisms with stacked Gol-
gi bodies such as the slime mold Dictyostelium discoideum, the
gymnamoebae like Acanthamoeba [24], as well as the flagellate
Breviata anathema (Fig. 3) [25]. This last organism is a recent addi-
tion to the Amoebozoa, based on concatenated gene phylogeny,
and may well represent a basal lineage in the supergroup [13].
2.3. Heterolobosea
Distantly related to the pelobionts, the Heterolobosea are an-
other group of amoeboflagellated protozoans that include both
aerobes and anaerobes. The most prominent is the ‘‘Brain-Eating
Amoeba” Naegleria fowleri, but most Naegleria species are globally
distributed and harmless free-living denizens of the soil [26]. The
heterolobosea are members of the supergroup Excavata and solidly
established as related to the jakobid flagellates and the euglenozoa,
such as the photosynthetic pond-dweller Euglena gracilis and Try-
panosoma brucei (Fig. 3), the causative agent of African sleeping
sickness [12,14]. All of these relatives have well-defined stacked
Golgi bodies [5], with the latter being a cell biological model for
the study of Golgi division [27]. Stephanopogon, which had for a
long time been considered incertae sedis (taxonomically unre-
solved), has been reliably placed as a heterolobosean [28].
2.4. Diplomonads and retortamonads
The diplomonads include organisms such as the fish pathogen
Spironucleus vortens, as well as Giardia, which is a major cause of
human diarrhoeal disease world-wide [29]. These obligate anaer-
Entamoeba
histolytica Dictyostelium Homo
discoideum sapiens
Drosophila
melanogaster
Breviata
anathema
Saccharomyces
cerevisiae
Pichia
pastoris
Schizosaccharomyces
pombe
Encephalitozoon
cuniculi
basal fungi
Monocercomonoides sp.
Trimastix
pyriformis
Giardia
intestinalis
 K. Mowbrey, J.B. Dacks / FEBS Letters 583 (2009) 3738–3745 3741

Fig. 3. Respectable citizens of protistology. Here are shown relatives of various ‘Golgi-lacking’ eukaryotes with micrograph of both the organism and their stacked Golgi
bodies. (A and B) Organismal and Golgi images modified from [20] of Pichia pastoris, the respectable relative of both S. cerevisiae and the microsporidia. Image modified from
[20], with permission and courtesy of B. Glick. (C and D) the relative of Entamoeba and the pelobionts, Breviata anathema, and its stacked Golgi body. Unpublished images
courtesy of G. Walker. (E and F) A colourful (VSG-tagged and DAPI stained) Trypanosoma brucei (unpublished image courtesy of S. Wang and M.C. Field) and its stacked Golgi
body (unpublished image courtesy of C. He). Trypanosoma is the sister taxa to the Heterolobosea. (G) The single cisternae Golgi body of Colpoda (unpublished courtesy of Denis
Lynn). (H and I) The apicomplexan Toxoplasma and its stacked Golgi, modified with permission from [72].
3742 K. Mowbrey, J.B. Dacks / FEBS Letters 583 (2009) 3738–3745
obes possess several unusual organelles such as a combined endo-
some/lysosome [30] and a mitosome [31]. Diplomonads are so
called because of their appearance of a bilaterally symmetrical cell
with two nuclei and two sets of flagella [29]. Retortamonads in-
clude anaerobic flagellates, such as Retortamonas, and Chilomastix,
found in some metazoan guts [32]. Each lineage has been proposed
to be the deepest branch in the eukaryotic tree and are often tou-
ted as such today. However, molecular phylogenetic data firmly
place them as sister taxa [33] embedded within the Excavata
[12]. Importantly, the free-living flagellate Carpediemonas membra-
nifera is firmly established as the basal sister to diplomonads and
retortamonads [34] in the taxonomic group Fornicata. Carpedie-
monas possesses a typical Golgi body [35]. Furthermore, the forni-
cates (as they are informally known) are the sister group to the
Parabasalia, which includes Trichomonas vaginalis, the causative
agent of the most prevalent, non-viral sexually-transmitted infec-
tion world-wide [36]. The Parabasalia are so named for their para-
basal apparatus, composed of a stacked Golgi body associated with
a striated cytoskeletal fiber. In fact, one of the most beautiful
examples of expanded Golgi body structure comes from the poly-
phyletic group of hypermastigote parabasalids, which possess
many Golgi stacks per cell and dozens of cisternae per stack (e.g.
[37]).
2.5. Oxymonads
The oxymonads are a group of obligately symbiotic or commen-
sal unicellular flagellates. All but one lineage are symbionts of ter-
mites or wood-eating cockroaches and have evolved spectacular
cellular features including cytoskeletal holdfast apparatuses and
motile axostyles [38]. Perhaps the most aberrant of the taxa dis-
cussed, oxymonads not only lack visible Golgi bodies but also vis-
ible mitochondria and peroxisomes, they have highly unusual
sexual cycles and some utilize alternative genetic codes [38,39].
Many also have extensive associations with symbiotic bacteria.
Based both on molecular sequence data [40] and ultrastructure
[32], the oxymonads are sister taxa to a free-living flagellate genus
called Trimastix, members of which possess typical stacked Golgi
bodies [41]. Together called the pre-axostyla [32], these lineages
are relatives to the Fornicata and Parabasalia [12,32,42], all encom-
passed in the supergroup Excavata.
2.6. Leukarachnion
Described in 1942, the amoeba Leukarachnion, was not one of
the originally implicated archaezoan taxa. A recent study of the
taxon described an amoeboid, multinucleate, heterotroph with
‘‘branching, ‘net-like’ pseudopodia” [43]. No obvious Golgi bodies
were observed. Analyses of both single gene and concatenated
gene trees with data from Leukarachnion placed it within the Stra-
menopiles, as a likely sister to the chrysophyte algae [43]. Although
the details of the internal relations of stramenopiles are not com-
pletely clear, there are very good examples of stramenopiles with
stacked Golgi bodies including the ‘water-mould’ oomycete patho-
gens (e.g. Phytophthora) [44], kelps and diatoms.
2.7. Apicomplexa
The final group of organisms putatively ‘lacking Golgi’ are par-
ticularly curious. The Apicomplexa include some of the most pre-
valent and deadly known human parasites. Plasmodium
falciparum causes cerebral malaria, with 500 million cases reported
annually and up to a million deaths per year [45], while Babesia
causes malaria-like symptoms in cattle [46]. Cryptosporidium
causes a gastrointestinal disorder that can be life threatening in
the immunocompromised. Golgi stacks are not visible in Babesia
[47], or Cryptosporidium [48], and were only recently characterized
in Plasmodium [49]. Here the Golgi bodies are composed of single
cisternae. Despite this unusual or absent Golgi complex, the Api-
complexa were never considered as a major ‘Golgi-lacking’ lineage
because of Toxoplasma gondii. This apicomplexan is thought to
have infected up to one third of the world’s population and can
cause fatal encephalitis in the immunocompromised [50]. It also
possesses a single, well-organized stacked Golgi body per cell
(Fig. 3) [27]. The Apicomplexa are nestled in the alveolate lineage
[15] along with the ciliates and the dinoflagellates which each have
stacked Golgi bodies. Interestingly, the Golgi complexes of the cil-
iate Colpoda (Fig. 3), and the more commonly known Tetrahymena,
are composed of one or a few cisternae, with hundreds of copies of
the organelle in the latter [51]. This means that these ciliate Golgi
bodies more closely resemble the organelle in Plasmodium than the
canonical looking organelle in Toxoplasma.
As seen in Fig. 2, each candidate ‘Golgi-lacking’ lineage is
embedded within a clade of organisms possessing stacked Golgi
bodies, implying secondary loss or morphological shift in each
case!
3. Golgi-associated genes are present in the genomes of all
‘Golgi-lacking’ organisms examined
Phylogenetic data and its implied conclusions are open to two
potential criticisms. Firstly, it can only speak to the primitive or de-
rived nature of Golgi bodies in each lineage, but not to whether the
organelle is actually present. Secondly, there is a logical loophole. If
one of those putatively ‘Golgi-lacking’ lineages were actually to be
the deepest divergent eukaryotic group, then that would imply the
lineage, and only that lineage, was in fact primitively missing Golgi
bodies. This, however, can only be true of one of the ‘Golgi-lacking’
groups. Because the various candidate ‘Golgi-lacking’ lines are
spread throughout the eukaryotic tree, and each is nested among
taxa with Golgi bodies, it is clear that most of them must be sec-
ondarily derived.
Nonetheless, the root of the eukaryotic tree has at various times
been proposed to lie on or near the branches containing some of
the ‘Golgi-lacking’ lineages [8,9], with some evidence still pointing
in that direction [52]. Alternatively, the Bikont–Unikont root,
which has Opisthokonta and Amoebozoa on one side, and Archaep-
lastida, Excavata and the SAR clade on the other was, up until re-
cently, a leading contender [16]. This placement of the root was
based on the correlation of molecular evidence putting unikonts
together in the tree, morphological evidence relating to an inter-
pretation of the arrangement of flagellar basal bodies in unikont
taxa, and the distribution of a fused metabolic gene for Dihydrofo-
late reductase and Thymidilate synthase (DHFR–TS), which was
observed to be fused in the bikonts sampled and present as sepa-
rate genes in bacteria and unikonts [16]. However, the placement
of the two-basal body possessing Breviata [13] and the biflagellate
apusumonads in the unikonts [53], as well as the recently de-
scribed extent of plasticity of the DHFR–TS fusion gene character
have undermined this idea considerably (discussed in [53]). There
is currently no clear resolution of where the eukaryotic root goes
and consequently what is the basal branch within the eukaryotic
tree. A different type of data is needed, one which speaks not only
to the history of the organelle, but whether it has been truly lost or
merely hidden in each given lineage.
The Golgi body, of course, is only one of several endomembrane
organelles that make up the endomembrane system, including the
endoplasmic reticulum (ER), the endosomes, lysosomes and the
plasma membrane. Transport of material among these organelles
involves packaging of cargo proteins into transport carriers of var-
ious morphologies, but usually thought of as vesicles. As almost
certainly described in more detail elsewhere in this volume, there
 K. Mowbrey, J.B. Dacks / FEBS Letters 583 (2009) 3738–3745
is a well-characterized set of protein machinery involved in the
formation, fusion, and specific targeting of these trafficked vesicles
[54]. Membrane-trafficking begins when a GTPase (Sar/Arf) marks
a site of nucleation on a donating organelle, and adaptors select
and concentrate proteins for transport, together establishing an in-
ner vesicle coat. Outer coat proteins then deform the membrane
and the vesicle buds away. Four different protein complexes
(named COPI, COPII, clathrin/adaptin and retromer) abide by this
model and mark discrete cellular locations [54]. When a vesicle
reaches its target organelle, docking and tethering lead to eventual
vesicle fusion. This process involves cellular machinery using pro-
teins such as Sec1/Munc18 (SM), Rab and SNARE [54,55], as well as
several heteromeric tethering complexes. One important aspect of
organization in the membrane-trafficking system is that many of
the proteins involved in vesicle trafficking are members of protein
families, with various family members performing the same func-
tion, but at discrete locations or transport pathways within the cell
[54]. Specificity of targeting is encoded in the combinatorial inter-
action of all of these factors [55].
The model of membrane-trafficking described above is based
primarily on studies from animal and yeast models [54]. However,
this represents a small fraction of eukaryotic diversity. Several
comparative genomic studies have shown that the core set of pro-
teins required for vesicle formation and fusion are conserved
across eukaryotic diversity (reviewed in [56,57]). This implies that
many mechanisms are universally applicable and that the machin-
ery evolved very early in eukaryotic evolution [56,57]. It also im-
plies a surprisingly complex set of membrane-trafficking
machinery present in the Last Common Eukaryotic Ancestor, with
at least all of the major protein families thought to be involved in
membrane-trafficking already on board over one billion years ago!
Because of the organelle-specific nature of many protein com-
ponents in the membrane-trafficking system, it is possible to de-
fine a ‘Golgi cohort’, a set of proteins that act primarily or
exclusively in trafficking from the Golgi body, such as coat proteins
like COPI, adaptor proteins like AP4 and components of the retro-
mer complex. It would also include components of the fusion
machinery that act to receive material at the Golgi body such as
the SNARE syntaxin 5, Rab6 or components of the tethering com-
plex COG. This Golgi cohort is conserved in organisms spanning
eukaryotic diversity [58–61], and so can be used as a signature
for the presence of a Golgi organelle, even in putatively ‘Golgi-lack-
ing’ lineages.
The first application of this idea came in 2003 [62]. Molecular
biology and phylogenetics was used to obtain Golgi cohort genes,
involved in both vesicle formation and fusion, from diplomonads,
entamoebids, heterolobosea and pelobionts [62]. Since that time
additional genes have been identified from oxymonads (CopG,
Table 1
Comparative genomic survey of Golgi cohort components in ‘Golgi-lacking’ taxa.
These results are based both on published literature [62,73–75] as well as our own
homology searching in publicly available genome databases for Babesia bovis (http://
www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) and Naegleria gruberi (http://geno-
me.jgi-psf.org/Naegr1/Naegr1.home.html) and EST collections for Mastigamoeba
balamuthi (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In cases where homology search-
ing was performed the following human queries were used: COPIG (NP_001091868),
AP1G (O43747), Vps26 (NP_001030337), Vps35 (NP_060676), Rab6 (NP_001003789),
Syntaxin 5 (NP_003155) and Trs23 (NP_057230). NI = not identified.
COPI AP1G Retromer Rab6 Syntaxin 5 TRAPPI
p p p p p p
Diplomonads
Oxymonads
p
NI
p
NI NI NI
p p p p p p
Entamoeba
p p p p p
Mastigamoeba NI
p p p p p
Microsporidia NI
p p p p p p
Heterolobosean
p p p p p p
Babesia
3743
Sly1 and the retromer component Vps35) [63]. Genome sequence
data are now available for Giardia intestinalis [64], the microsporid-
ian Encephalitozoon cuniculi [65] and Entamoeba histolytica [66] and
these encode diverse proteins in the Golgi cohort. As well, such
genes have been found within the genome of the heterolobosean
Naegleria gruberi (unpublished data Dacks, Field and Dawson). Ta-
ble 1 summarizes published accounts of Golgi cohort genes for
‘Golgi-lacking’ taxa as well as homology searching that we per-
formed for Babesia, and Mastigamoeba. With the exception of
retortamonads and Leukarachnion, for which no molecular data
are available, in all cases genes of the Golgi cohort have been iden-
tified. This argues against the primitive absence of Golgi bodies or
that the organelle has been completely lost. Rather it suggests that
a Golgi homologue is present in each of these organisms, and that
the morphology has shifted to some unrecognizable morphology.
4. Golgi homologues found in ‘Golgi-lacking’ taxa
Of course, the most convincing evidence is the localization of
proteins in the Golgi cohort to organelles within the cell. This not
only demonstrates expression of the gene and functional homology
of the protein, but reveals the variation of morphology that is ob-
served in Golgi bodies.
Giardia trophozoites must sort and secrete colossal amounts of
cyst wall proteins (CWPs) in order to form the dense extracellular
matrix of the cyst wall. This task, in Giardia cells, is the responsibil-
ity of encystation-specific vesicles (ESVs). Evidence bolstering sup-
port for ESVs as Golgi body homologues includes the upregulation
of GlcNAc- and GaINAc-transferase at end stages of encystation,
and the incorporation of the Golgi lipid marker NBD-ceramide into
ESV membranes of encysting cells but not those of trophozoites
([67] and references therein). The exact mechanism underlying
the secretion of CWPs by ESVs is unknown, with proposals ranging
from direct fusion of ESVs with the plasma membrane to secretion
of CWPs from numerously dispersed ESVs in smaller portions. Re-
cent evidence suggests that ESVs may represent a mixed popula-
tion of compartments representing various stages of ER exit site/
Golgi cisternae, with material transferred between them by tubular
rather than vesicular carriers [67].
Critical to the parasite’s pathogenicity, E. histolytica synthesizes
an array of glycoproteins, lectins and glycolipids. However, in
eukaryotes, these glycoconjugates are produced by the ER and Gol-
gi body. This prompted many researchers to closely scrutinize the
secretory system of the protist, providing the results that Golgi-like
elements are, in fact, present in E. histolytica. Ultrastructural cryo-
fixation and cryosubstitution studies have revealed flattened cis-
ternae in the cytoplasm similar to those of most eukaryotes [68].
Other studies have demonstrated antibodies against mammalian
Golgi-cohort proteins such as ARF and e-COP could be disrupted
in labeled vesicles of trophozoites by Brefeldin A, a common dis-
rupter of Golgi body structure [69].
Perplexed by the state of Golgi bodies, or lack there of, in Plas-
modium falciparum, many studies have sought to characterize the
state of Golgi-defining proteins in the parasite. One such example
is the Golgi reassembly stacking proteins (GRASPs). GRASP proteins
are one of the few prominent Golgi markers conserved across
eukaryotic diversity, involved in cisternal signaling and stacking,
and, as such, have been utilized in examinations of the architecture
and dynamics of protozoan Golgi bodies. Evidence suggests that
PfGRASP is transcribed and expressed throughout the asexual life-
cycle of P. falciparum, confirmed by western blot analysis of trans-
genic parasites [49]. Furthermore, immunofluorescence assays
using antibodies against marker proteins for the Golgi, such as
anti-PfERD2, demonstrate colocalization to PfGRASP-GFP, which
confirms the identity of the PfGRASP compartments [49]. These
findings support PfGRASP as an important and valid Golgi body
3744 K. Mowbrey, J.B. Dacks / FEBS Letters 583 (2009) 3738–3745
marker, and substantiate notions of P. falciparum as possessing
Golgi-like properties.
5. Conclusions and questions
In all of the lineages examined, homologues of Golgi cohort
genes have been found, and whenever tested these have been use-
ful in identifying putative Golgi organelles. Since there is no strong
evidence that the eukaryotic root lies directly on any of the candi-
date lineages, it seems that there are in fact no ‘Golgi-lacking’
eukaryotes. The Last Common Eukaryotic Ancestor very likely did
possess a Golgi body, and it was very likely to have been stacked!
Even if the root were to lie on one of those candidate lineages,
and the ancestor was unstacked, the dispersal of the organisms
in question across the eukaryotic tree means that Golgi stacking
has been lost a minimum of eight separate times (Fig. 2). This
count could well change depending on the resolution of organisms
within the Apicomplexa, where the simplest explanation is a min-
imum of two separate instances of Golgi unstacking.
Nonetheless, there are important questions about the evolution
of Golgi bodies left to be addressed. Firstly, will additional micro-
bial eukaryotes lacking Golgi stacks be described? New environ-
ments are being explored and new organisms are being
described all the time. This is in addition to the 200 organisms
described at the light microscope level, whose phylogenetic affin-
ity are currently undetermined [15].
Secondly, where does the root of eukaryotes actually lie? This is
one of the major questions facing the study of eukaryotic evolution
and it is not necessarily clear from where the answer will come.
Fusion gene characters are not the Holy Grail once thought; having
not yet yielded data that is either internally consistent or congru-
ent with other characters, once more than a few taxa are examined
[53]. Concatenated gene phylogenies still do not appear to be able
to completely overcome pervasive artefacts from the variations in
rate of evolution among taxa [70]. Perhaps genomic sampling of
slowly-evolving representatives from the major groups, and more
tractable application of sophisticated models of sequence evolu-
tion will point the way.
Finally, despite our emphasis here on eukaryotes with un-
stacked Golgi bodies, the vast majority of organisms do possess
stacked Golgi organelles of one flavour or another. And so, while
we can conclude that Golgi unstacking has occurred multiple times
in eukaryotic history, we do not know why. The obvious patterns
do not seem to hold: there are aerobic and anaerobic representa-
tives, free-living organisms and parasites. There appears to be
some correlation with the degree of glycosylation, since organisms
such as Giardia and Plasmodium possess fewer glycosylases than
relatives possessing Golgi stacks [71]. However, it is not clear
how tight this pattern is, or even whether this is causative or
merely correlative.
Any birthday is a time for reflection about the past and specu-
lation as to what the future holds. On the 110th anniversary since
the discovery of the Golgi body, new genomes from diverse
eukaryotes are being sequenced, more powerful molecular evolu-
tionary methods are being developed, and new microbial eukary-
otes are being described. It is an exciting time for the field of
evolutionary cell biology and the study of the Golgi body.
Acknowledgements
Images were generously provided by Mark Field and Susan
Wang (Trypanosoma brucei), Lillian Fritz-Laylin (Naegleria), Scott
Dawson and Susan House (Giardia), Ben Glick (Pichia), Brian Lean-
der (Stephanopogon), Vladimir Hampl (Monocercomonoides) and Gi-
selle Walker (Breviata, Mastigamoeba), as well as Cynthia He and
Lawrence Pelletier (Toxoplasma and Trypanosoma Golgi). We would
like to thank Giselle Walker and Paul Lapointe for critical reading
of the manuscript. This work was funded by NSERC grant
G121211166 and start-up funds from the Faculty of Medicine at
the University of Alberta to J.B.D.
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