Secretory pathway of cellulase

12 Pages·2013·0.86 MB·English

YanandWuBiotechnologyforBiofuels2013,6:177
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REVIEW Open Access
Secretory pathway of cellulase: a mini-review
Shaomin Yan1 and Guang Wu1,2*
Abstract
Cellulase plays an important role inmodern industry and holds great potentialin biofuel production.Many different
types oforganismsproduce cellulase, which go through secretory pathways to reach the extracellular space, where
enzymatic reactions take place. Secretory pathways invarious cellshave been the focus of many research fields;
however, there are few studieson secretory pathways of cellulases inthe literature. It is therefore necessary and
important to review thecurrentknowledgeon the secretory pathways of cellulases. Inthis mini-review, we address
thesubcellular locations ofcellulases indifferent organisms, discuss the secretory pathways of cellulases indifferent
organisms, and examinethesecretory mechanisms of cellulases.These sections start with a description of general
secreted proteins, advance to the situation of cellulases, and end with theknowledgeof cellulases,as documented
inUniProt Knowledgebase (UniProtKB). Finally, gaps inexisting knowledge are highlighted,which mayshed light
onfuture studies for biofuel engineering.
Keywords: Cellulase, Secretory pathway, Subcellular location,Secretory mechanism,UniProtKB
Introduction thesecretion ofa singleenzyme,but agroup ofproteins.
Cellulase is a word by combining cellul(ose) with -ase, For example the secretion of proteins in filamentous
and appeared around 1900 to 1905 [1], whereas the fungi may reach up to 20 g/L of extracellular medium
word cellulose appeared between 1745 and 1755 [2]. [10] and the secretion of alkaline extracellular protease
Cellulase belongs to EC 3.2.1.4 and catalyzes the endo- byYarrowia lipolytica reaches 1g/L[11].
hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, A more complete knowledge of the cellulase secretory
lichenin, and cereal beta-D-glucans [3]. In a broader pathwaywillnotonlyhelpustounderstandthistheoret-
sense, exoglucanases (EC 3.2.1.74 and EC 3.2.1.91) and ical topic, but will also help the selection of organisms
β-glucosidases (EC 3.2.1.21) are also classified as cellu- which most efficiently secrete cellulases, selection of cel-
lases [4,5]. In the modern world, cellulase has many ap- lulases which already exist in the extracellular matrix,
plications in industry because of the wide existence of and selection of organisms whose secretory pathways
cellulose, lichenin, and cereal beta-D-glucans [6]. Many have less frequent mutations, and so on. This is import-
different types of organisms can produce cellulases, for ant because cellulose is the most abundant component
example bacteria, fungi, protozoa, and some animal spe- of plant biomass and has wide industrial applications,
cies, including termites and crayfish, which produce with a very promising prospective in the biofuel indus-
their own cellulases and differ substantially from those try. Indeed, the conversion from biomass to biofuel can
oftheirindigenousmicroflora[7]. be divided into pretreatment, hydrolysis, fermentation,
Cellulase plays amainlycatalyticrole intheextracellu- and distillation/evaporation [12], with cellulases involved
lar matrix where the enzymatic reaction takes place inthewholeprocess of hydrolysis.
[8,9]. Therefore, cellulases are produced from cells A typical secretory pathway in a cell is generally com-
through a certain pathway, which may not be limited to posed ofatleasttwo components, endoplasmicreticulum
and Golgi apparatus, and a typical cell generally has two
endomembrane systems, one for incoming traffic and the
*Correspondence:[email protected]
1StateKeyLaboratoryofNon-foodBiomassEnzymeTechnology,National other for outgoing traffic [13]. A protein generally under-
EngineeringResearchCenterforNon-foodBiorefinery,GuangxiKey goes the following process to be ready to move out of a
LaboratoryofBiorefinery,GuangxiAcademyofSciences,98DalingRoad, cell: protein biosynthesis, translocation to endoplasmic
Nanning,Guangxi530007,China
2DreamSciTech,Apt207,Zhencaili26,ZhujiangRoad,HexiDistrict,Tianjin reticulum, attachment of N-glycan, glycoprotein folding,
300222,China
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waiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwise
stated.
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N-andO-glycosylation,transportationtoGolgiapparatus, melanosome, microsome, mitochondrion, nucleus, per-
proteinsortingandformationofsecretoryvesicles,vesicle oxisome, plasma membrane, spindle pole body, synapse,
budding, transport, and vesicle fusion with the plasma and vacuole [19]. A human cell includes 12 subcellular
membrane [13,14]. Those components form the general locations: centriole, cytoplasm, cytoskeleton, endosomal,
concept that eukaryotic cells use for the endoplasmic endoplasmicreticulum,Golgiapparatus,lysosome,mito-
reticulum-to-Golgimembranesecretorypathway. chondria, nucleus, peroxisome, plasma membrane, and
Various secretory cells exist, which include endocrine synapse. A plant cell includes ten subcellular locations:
cells, exocrine cells, and immune cells [15-17]. For in- cell wall, chloroplast, cytoplasm, endoplasmic reticulum,
stance, in response to ultraviolet exposure, melanocytes mitochondria, nucleus, peroxisome, plasma membrane,
synthesize melanin to form melanosomes and are then plastid, and vacuole. A Gram-positive bacterium in-
transferred to keratinocytes, which is considered to be a cludes four subcellular locations: cell wall, cytoplasm,
specialized type of secretion [18]. However, it is not yet periplasm, and plasma membrane; and a Gram-negative
known whether the cellsthat secrete cellulases belong to bacterium includes seven subcellular locations: cyto-
such specific secretory cells. Thus, there are a series of plasm, fimbrium, flagellum, inner membrane, nucleoid,
questions relating to the secretory pathway of cellulases outermembrane,andperiplasm[20].
that need answers by reviewing the literature, including: A protein’s subcellular location can lead its specific
1) Can we classify the cells that produce cellulases as function and critically influences cell functionality. For
secretory cells? 2) Where are the subcellular locations example the redox potential, which is maintained by en-
for cellulases? 3) Does a cellulase use the endoplasmic zymes such as oxidase and endothelial nitric oxide syn-
reticulum-to-Golgimembranepathwayforsecretion?4)Is thase [21], in a eukaryotic cell is proposed from most
a cellulase processed within the Golgi apparatus soluble? oxidizing to most reductive, as follows: mitochondrion>
5) Does a cellulase adopt a different secretory pathway nucleus>cytoplasm>endoplasmic reticulum>extracel-
from the common secretory pathway? 6) What are the lular [22]. Needless to say, the difference inredox poten-
special characteristics of cellulases for their secretion? tial has a direct impact on the cellular signaling system.
Thereafter,wealsohopetousethereviewedknowledgeto On the other hand, an enzyme that has several subcellu-
examine cellulases documented in UniProt Knowledge- larlocationscouldhavedifferent concentrations inorder
base(UniProtKB)[5],whichwasreleasedon24July2013 tofunctiondifferentially.
andincluded4,101cellulaseswithaccessionnumbers.
In this context, it is necessary to address the secretary Subcellularlocationsofcellulasesindifferentorganisms
pathway of cellulase in this mini-review. With rapid ad- The subcellular locations of cellulases are a topic that
vances in research facilities and technologies, the focus has been studied for several decades, with different tech-
of research shifts rapidly across different levels. For ex- niques available at given times. As early as the 1970s,
ample current research is heavily based on the genetic the subcellular locations of buffer-soluble cellulase and
level, which would have been impossible several decades buffer-insoluble cellulase from auxin-treated peas were
ago. A balanced review will not only address the results studied [23], and the authors found that buffer-soluble
obtained from modern techniques but will also uncover cellulase was localized at the inner surface of the cell
the results obtained from earlier techniques, and play a wall while buffer-insoluble cellulase was localized in the
complementary role to the understanding of the given endoplasmic reticulum. This finding partially answered
problems. thequestion ofwhethercellulaseissoluble,whichisalso
confirmed by the fact that five types of secretory path-
Location of cellulases in different organisms ways in Gram-negative bacteria address soluble proteins
Subcellularlocationsindifferentorganisms [24]. Later on, the location of cellulase was suggested to
In general, microorganisms cannot be considered as spe- be in cytoplasmic vesicles with 150 nm diameters by
cialized secretory cells, such as the cells that secrete in- isolation of cellulase-containing membranes of Achlya
sulin, sweat, and so on, because secretion of proteins ambisexualis Raper, and it was also found that IDPase,
only accounts for a fraction of activities of microorgan- ATPase, UDPG transferase, and sedimentable carbohy-
isms. However, the secretion of proteins in microorgan- drate were locatedinsimilar places[25].
isms still requires a series of operations between various Studies on fungi have demonstrated that the Golgi ap-
subcellular locations, from synthesizing proteins to paratus in fungi cells do not have a stacked appearance
transportingthemintothe extracellular matrix. and some fungi have many individual endoplasmic
A eukaryotic cell includes the following 21 subcellular reticulum-associatedsaccules[25-27].Cellulasewasfound
locations: acrosome, cell wall, centriole, chloroplast, cya- to be located in the vesicles that derived from endoplas-
nelle, cytoplasm, cytoskeleton, endoplasmic reticulum, micreticulum,hadribosomes,andattachedtotheoutside
endosome, Golgi apparatus, hydrogenosome, lysosome, surfaceofthemembrane[28].
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β-glucosidases (EC 3.2.1.21) are grouped according to percentage is higher than those in Gram-negative and
location, including intracellular, cell wall-associated, and Gram-positive bacteria.Thismayimply thatthesecretory
extracellular [29]. For example β-1,4,-D-endoglucanase pathway in eukaryotic cells is more efficient than in
was found to be located on the outside surface of Prevo- Gram-negative and Gram-positive bacteria. It is not ne-
tellaruminicola[30],which isa starch-degrading bacter- cessary that cellulases documented in UniProtKB are lo-
ium but utilizes water-soluble cellodextrins [31], and of cated in a single subcellular location, for example a
which some strains have considerable carboxymethyl cellulasefromStreptomycesreticuli[UniProt:Q05156]was
cellulase(CMCase)activity[32-35]. found to exist in both mycelium-associated and extracel-
Enzymological studies have shown that bacterium has lularforms.
both extracellular and cell-bound endoglucanase activ-
ities, of which up to 80% were found in the extracellular Secretory pathways in different organisms
fluid in the stationary-phase of cellulose-grown cultures. Secretorypathwaysingeneral
In Fibrobacter succinogenes subspecies succinogenes S85, In the late 1980s, the term autotransporter was coined
a small part of endoglucanase was found in the periplas- for secreted proteins as a type of secretory pathway in
mic fraction while a large part was found in the cyto- Gram-negative bacteria, because Gram-negative bacteria
plasmic and membrane fractions [36]. Groleau et al. have two asymmetric biological membranes while
demonstrated that the majority of the cell-free cellulase Gram-positive bacteria have only one. To date, seven
was associated with sedimentable membrane fragments, typesofsecretory pathwayshavebeendefinedfor secret-
and the rest was obtained from a fraction of low- ing soluble proteins in Gram-negative bacteria [45,46].
molecular-mass that was approximately 45 kDa and Type I secretory pathway includes an oligomeric com-
nonsedimentable protein aggregates that were larger plex composed of an inner membrane ATP-binding cas-
than 4 × 103 kDa [37-39]. Also, three separate endoglu- sette exporter, a membrane fusion protein, and an outer
canases designated EG1, EG2 [40], and EG3 [41], an membrane homologue [47,48]. Type II secretory path-
extracellular Cl-stimulated cellobiosidase [42], and a way, which has an alternative name as the general
periplasmic cellodextrinase [43,44] were purified and secretory pathway, presents a two-step process: 1) pro-
characterized. The cellobiosidases were found to associ- teins are moved across the inner membrane through the
atewithactivelygrowingcellsinculture [44]. Sec system; and 2) proteins are moved across the outer
membrane[49,50].TypeIIIsecretorypathwayisahighly
SubcellularlocationsofcellulasedescribedinUniProt regulated channel through both the inner and outer
Knowledgebase(UniProtKB) membranes forming a needle-like structure [51-53].
Of 4,101 cellulases including 741 fragments from Type IV secretory pathway involves the conjugative
UniProtKB, 121 cellulases are evidenced at protein level, transfer of DNA as well as nucleoprotein complexes,
306 cellulases are evidenced at transcript level, 217 cellu- and is further divided into type IVa and IVb according
lasesareinferred fromhomology,and 3,457cellulasesare to sequence homology [53-55]. Type V secretory path-
predicted. However, only 85 cellulases are documented way has the simplest secretion apparatus and represents
withtheirsubcellularlocations.InTable1,atproteinlevel, a large family of protein-translocating outer membrane
75%(24/32)ofcellulasesareannotatedasasecretedform, porins [56]. TypeVI secretory pathway is a newly discov-
that is, they were found in the extracellular matrix. At ered pathway [57,58], which spreads in Gram-negative
transcript level, 30% (3/10) of cellulases are annotated bacteria, and plant and human pathogens as well [59-61].
with their location in the nucleus, that is, these cellulases For Mycobacterium, there is the type VII secretory path-
should be in eukaryotic cells. Cellulases inferred from way, which also exists in Gram-positive bacteria but to a
homology resulted in 79.49% (31/39) in secreted form, far less extent [62-64].However, typeVII is not related to
that is, secreted cellulases are highly homologous rather cellulaseaccordingtothecurrentknowledge.
thanheterologous.InTable2,86.27%(44/51)ofcellulases Gram-positive bacteria have fewer subcellular locations
are annotated as secreted in eukaryotic cells, and this and simpler membranes than those in Gram-negative
Table1SubcellularlocationsofcellulasesinUniProtKBaccordingtothesequencestatus
Sequencestatus Cellmembrane Cytoplasm Nucleus Periplasm Secreted Total
Evidenceatproteinlevel 5(15.63%) 1(3.13%) 0 2(6.25%) 24(75%) 32
Evidenceattranscriptlevel 0 0 3(30%) 0 7(70%) 10
Inferredfromhomology 2(5.13%) 6(15.38%) 0 0 31(79.49%) 39
Predicted 0 1(25%) 2(50%) 0 1(25%) 4
Total 7(8.24%) 8(9.41%) 5(5.88%) 2(2.35%) 66(74.12%) 86
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Table2SubcellularlocationsofcellulasesinUniProtKBaccordingtocelltypes
Cell Cellmembrane Cytoplasm Nucleus Periplasm Secreted Total
Eukaryota 2(3.92%) 0 5(9.8%) 0 44(86.27%) 51
Gram-negativebacteria 3(15%) 3(15%) 0 2(10%) 11(55%) 20
Gram-positivebacteria 2(15.38%) 4(30.77%) 0 0 8(61.54%) 13
Undeterminedbacterium 0 1(100%) 0 0 0 1
Total 7(8.24%) 8(9.41%) 5(5.88%) 2(2.35%) 63(74.12%) 85
bacteria,anddonothaveadedicatedapparatusforfolding primary glycosylation, folding and quality control, and
secreted polypeptides. However, this by no means implies vesicle-mediated secretion are similar from yeasts to
that Gram-positive bacteria have fewer secretory path- highereukaryotes.However,recentresearchhasindicated
ways. In fact, several secretory pathways are found in that significant functionaldifferences existbetween yeasts
Gram-positivebacteria:secretion(Sec),twin-argininetrans- andmammaliancells[77].
location (Tat), flagella export apparatus (FEA), fimbrilin- The secretory pathway in Archaea has been mainly
protein exporter (FPE), hole forming (holin), and the studied using genomic sequencing data, and compared
WXG100 secretion system (Wss) [65]. However, the Sec against the secretory pathways in bacteria and Eucarya
secretorypathwayisthemostrelevanttoproteinsecretion [78,79]. It was found that the secretory pathway in Ar-
because a Sec-type pathway is used in an important chaea is similar tothe Sec systemin bacteriaand Eucarya
human pathogen, Streptococcus pyogenes, which proceeds [80] because most Archaea have a homologue of CsaA, a
through a single microdomain [66], and the involvement protein involved in protein targeting in Bacillus subtilis
of Sec-type pathways [67-69] is more likely to be SecA [81,82], although they have a lipid monolayer instead of a
because secretion of cytotoxins can be inhibited by the phospholipidbilayer.
SecAinhibitorsodiumazideinBacilluscereus. Table 3 summarizes the general secretory pathways in
In mammalian cells, the vesicles that contain synthe- different organisms. Although secretory pathways are
sized proteins travel along microtubules from the rough termed and classified with a small number of appara-
endoplasmic reticulum towards the cis-Golgi or an tuses in cells, numerous different types of proteins have
endoplasmic reticulum-to-Golgi intermediate compart- been identified as major components for the construc-
ment (ERGIC). It is noted that a number of proteins tion of secretory pathways, including membrane traffic
never enter into this pathway, and these proteins are and protein secretion [14]. The secretion of proteins is
generally considered to be involved in cell survival, im- also called protein production. However, the secretory
mune surveillance, and tissue organization with funda- pathway is not limited to secreted proteins produced by
mental importance. Therefore, it is proposed that at least the organism itself, but also involves secreted foreign
the secretion of these proteins can be classified according substances, for example drugs and their metabolites
to whether the proteins go through a non-vesicular [83], and secreted cytotoxic substances, such as orphan
secretory pathway or a vesicular secretory pathway. For granzymes[84].
the non-vesicular secretory pathway, two types have been
defined: type I is a self-sustained protein translocation Secretorypathwaysofcellulase
across plasma membranes, and type II is an ATP-binding A single organism is not limited to secretion of a single
cassette transporter-based secretion. For the vesicular type of cellulase but several different types, which work
secretory pathway, two types have also been defined: type in a synergistic manner. This is similar to the secretion
IIIisanautophagy-basedsecretion,andtypeIVcomprises of hemicellulase, for example aerobic fungi Trichoderma
proteins that bypass the Golgi apparatus to transport to reesei and Aspergillus niger secrete 8 and 12 hemicellu-
theplasmamembrane.However,typesI,II,andIIIarein- lases in high concentrations, respectively [85]. Fungi,
volved in the secretion of cytoplasmic proteins while type such as T. reesei and A. niger, produce large amounts of
IVconsistsofintegralmembraneproteins[70]whichusu- extracellular cellulolytic enzymes, whereas some strains,
allyhavedistinctregionsofhydrophobicity[71-73]. including bacteria mainly from the class Clostridia, such
A typical secretory pathway in a eukaryotic cell begins as Clostridium cellulovorans [86-88] and Clostridium
withbudding from the endoplasmic reticulum to forming thermocellum [89,90], a few anaerobic fungi [91,92], and
thecoatproteincomplexII(COPII)vesicleseitherfullyor Eisenia fetida [93], mostly produce cellulolytic enzymes
partially uncoated [74]. In yeast, protein secretion has in a multienzyme complex called cellulosome, which is
been well-studied because yeast has cell wall synthesizing associated withthedegradingcellwall[94,95].
enzymes [75,76]. It has been suggested that the essential Secreted proteins can include homologous and heter-
functions through the endoplasmic reticulum membrane, ologous proteins, for example a typical cellulosome can
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Table3Listofgeneralsecretorypathwaysindifferentorganisms
Organism Secretorypathway Description
Gram-negativebacteria TypeIsecretorypathway Anoligomericcomplexcomposedofaninnermembrane
ATP-bindingcassetteexporter,amembranefusionprotein,
andanoutermembranehomologue.
TypeIIsecretorypathway,alsoknown Atwo-stepprocess:1)proteinsaremovedacrosstheinner
asthegeneralsecretorypathway membranethroughtheSecsystem,and2)proteinsare
movedacrosstheoutermembrane.
TypeIIIsecretorypathway Ahighlyregulatedchannelthroughboththeinnerand
outermembranesforminganeedle-likestructure.
TypeIVsecretorypathway InvolvesconjugativetransferofDNAandnucleoproteincomplexes.
TypeVsecretorypathway Alargefamilyofprotein-translocatingoutermembraneporins.
TypeVIsecretorypathway Formsatransenvelopeapparatus.Italsoexistsinplant,animal,
andhumanpathogens,andenvironmentalstrains.
TypeVIIsecretorypathway ExistsmainlyinMycobacteriumandGram-positivebacteriatoa
smalldegree.
Gram-positivebacteria Sec-typepathways InvolvesSec-typesignalpeptides.
Mammaliancells Non-vesicularsecretorypathway TypeIisaself-sustainedproteintranslocationacrossplasmamembranes.
TypeIIisanATP-bindingcassettetransporter-basedsecretion.
Vesicularsecretorypathway TypeIIIisanautophagy-basedsecretion.
TypeIVcomprisestheproteinsthatbypasstheGolgiapparatusto
transporttotheplasmamembrane.
Eukaryoticcells Buddingfromendoplasmicreticulumtoform Essentialprocessesaresimilarfromyeaststohighereukaryotes.
thecoatproteincomplexII(COPII)vesicles
Archaea SimilartoSec-typepathways MostArchaeahaveahomologueofCsaA.
be composed of 20 or more different cellulolytic/hemi-
cellulolytic enzymes in anaerobic bacteria, while in
anaerobic fungi, for example Neocallimastix frontalis
and Piromyces, a cellulosome-type complex includes at
least six or ten polypeptides [92,95,96]. Accordingly, an
18-subunitproteincomplexhasbeenengineeredtomul-
tienzyme structures called rosettasomes [97]. The mod-
eling of cellulosome self-assembly showed that the shape
and modularity were the dominant factors influencing
the cellulosomeassemblyprocess[98].
SecretorypathwaysofcellulasedescribedinUniProtKB
Of the 4,101 cellulases in UniProtKB, 133 cellulases
come from Archaea, 2,799 from bacteria, 928 from
Eukaryota,andonlytwo from viruses,andtheremaining
239are unclassified (upper panel of Figure 1). Therefore,
a vast majority of cellulases in UniProtKB come from
bacteria and Eukaryota, of which further classification is
listed in the lower panel of Figure 1. Since the secretory
pathways in bacteria are defined according to Gram-
negative and Gram-positive, excluding undetermined
bacteria, there are 1,210 Gram-positive bacteria and
1,365 Gram-negative bacteria in UniProtKB. Therefore,
when addressing cellulases from UniProtKB, it is likely
that a cellulase will follow one of seven secretory path-
Figure1Summarydataoforganismssecretingcellulases
ways defined in Gram-negative bacteria. Indeed, cellu-
documentedinUniProtKB.
lase [UniProt:Q38890] is annotated as a single-pass type
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II membrane protein, but from a plant cell. The type V system should be considered, which works for the first
secretory pathway is unlikely to exist in bacteria secret- part of the working mechanism of the type II secretory
ing cellulases, because type V operates on the autotran- pathway. Proteins with N-terminal signal peptides are
sporter. A search for autotransporter proteins in understood to be secreted by the Sec-dependent path-
UniProtKB does not reveal any cellulases belonging to way [49]. SecA is an ATP-dependent motor protein, but
the autotransporter; however, Yersinia pestis does have is associated with the SecYEG complex on the cytoplas-
nucleotide sequences checkable for autotransporter mic side and promotes the access of the chaperone-
search. Therefore, 124 cellulases from Y. pestis may be assisted substrate proteins to the inner membrane
considered as candidates for the type V secretory path- SecYEG complex [103]. SecB, a secretion-dedicated
way inGram-negative bacteria. chaperone, can recognize cytosolic protein precursors
[104,105]. Both SecE and SecG are auxiliary molecules
Secretory mechanisms for the translocase activity, while SecY is absolutely ne-
Secretorymechanismsingeneral cessary [106]. SecYEG is a translocase complex in the
It is estimated that a third of proteins synthesized within cytoplasmic or inner membrane and uses a substrate
a cell come from the endoplasmic reticulum [14]. The that can be targeted by a G protein precursor [107-110].
secretion is triggered by signal peptide-dependent pro- Additional observations of type II have been found in
tein translocation into the lumen of the endoplasmic Erwiniacarotovora,Erwiniachrysanthemi(Echr),K.oxy-
reticulum, followed by vesicular transport of secretory toca, Pseudomonas aeruginosa, Xanthomonas campes-
cargo to the Golgi apparatus membranes, and thereafter tris,andAeromonashydrophila [45,111].
to the cell surface. In addition, the proteins known as For the type III secretory pathway, the mechanism is
tethering factors are needed to build an initial connec- well-characterized in Salmonella and Yersinia. It has a
tion between vesicles and the destination membrane complex apparatus, which is also named injectisome or
[74,99]. In this view, for many proteins that are secreted molecular needle, because assembled proteins form a
into the extracellular matrix, their subcellular locations highly regulated channel through both the inner and
are dynamically regulated by various mechanisms. For outer membranes in a needle-like structure. This appar-
example, although the secretory pathway in Gram- atus secretes proteins not only into the extracellular mi-
negative bacteria is related to two membranes, seven lieu but also directly into a target eukaryotic cell. In fact,
types operate indifferent mechanisms. many Gram-negative plant and animal pathogenic bac-
For the type I secretory pathway, the mechanism teria use this system as a molecular syringe to inject ef-
shown in Escherichia coli with HlyA toxin is signal fector proteins directly into the host cell. The regulation
sequence-independent without free periplasmic inter- of this secretory pathway is mediated by a bacterial
mediate [45,100]. The secretion is undertaken by a translocon, and there are several putative secretion
translocator spanning the cell envelope with three pro- translocon proteins that have been identified with struc-
teins: 1) a specific outer membrane protein, 2) a cyto- turalsimilarity[51].
plasmic membrane protein, ATP-binding cassette, and For the type IV secretory pathway, the mechanism
3) a cytoplasmic membrane protein, membrane fusion/ seems to be more related to clinical settings, where bac-
adaptor protein. The secreted proteins do not require a terial conjugation is problematic because it leads to a
cleaved C-terminal signal, because this signal sufficiently rapid dissemination of antibiotic resistance genes and
recognizes the ATP-binding cassette protein leading to other virulence traits among bacterial populations. This
the assembly of the functional transenvelope complex. pathway delivers effector molecules, including DNA and
[101].Itisestimatedthatthetransportofpolypeptidesup protein substrates such as the pertussis toxin, as well as
to900kDaacrossthecellenvelopetakesafewseconds. monomericproteinssuchasRecA,toinfluenceeukaryotic
For the type II secretory pathway, the mechanism target cells during infection. Thus it is also known as a
operates at two different locations, as shown in studies macromolecular transfer system and found in several
on the pullulanase enzyme from Klebsiella oxytoca. The pathogens of plants and mammals, including Agrobacter-
first mechanism involves the Sec system to transport ium tumefaciens, Bordetella pertussis, Helicobacter pylori,
proteins across the inner membrane, while the second and Legionella pneumophila. Two subclasses have been
involves the transport of effector molecules across the definedbasedonsequencehomology[54]:1)typeIVare-
outer membrane, which works for a specific secretion ferstothemachineryassembledfromVirBhomologuesof
apparatus,secreton,composedof upto16 differentgene A.tumefaciens,and2)typeIVbreferstothefunctionalse-
products and at some stage spanning the entire cell en- cretion system assembled from Tra homologues of the
velope [49,50], and general secretion proteins (GspD, InclColIb-P9plasmidofShigellaflexneri[55].
GspE, and GspF) help the second mechanism [102]. For the type Vsecretory pathway, the mechanism was
When examining the type II secretory pathway, the Sec first described for the IgA1 protease produced by
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Neisseria gonorrhoeae [112]. This pathway has the sim- trigger the secretion of endoglucanases as shown in an
plest secretion apparatus, is composed of the largest fam- early study by McGavin et al. [36]. Extracellular concen-
ily of protein-translocating outer membrane porins in tration of chlorine can also stimulate the secretion of
Gram-negative bacteria [56], and requires the protein cellobiosidase [43]. It has been shown that the secretion
precursors to have three functional domains: 1) the N- systemforcellulaseinE.carotovorasubspeciescarotovora
terminalleaderinitiatingtheinnermembranetransportof (Ecc) belongs to type II of Gram-negative bacterium, and
the precursor, 2) the mature part of the protein forming is highly homologous in a wide range of bacteria
the extracellular functional domain, and 3) a C-terminal [45,111,121]. As early as the 1980s, the issue of whether
helperdomainthatisessentialforextracellularsecretion. synthesisofendoglucanaseactivitywasregulatedbyacar-
For the type VI secretory pathway, the mechanism bonsourcewasstudiedinC.thermocellum[122-126].
seems to be adapted by individual bacterial species to With regard to anterograde and retrograde transport,
interactwithotherprokaryotes,eukaryotes,orboth[113], the secretory pathway was depicted with the following
andthestructureformsatransenvelopeapparatus[114]. steps: 1) N-glycan biosynthesis and transfer to aspara-
The aforementioned mechanisms are related to only gine in normally glycosylated target proteins, 2) endo-
how a protein is secreted, while the mechanisms that plasmic reticulum-to-Golgi network anterograde and
regulate each secretory pathway are far more compli- retrograde transport, 3) Rab GTPases and interacting
cated. This is not only because current research focuses factors mediate this process, 4) process related to micro-
on the genes, which produce protein regulating secre- tubules, and 5) the endoplasmic reticulum-associated
tions, but also because those regulatingproteinsare sub- degradation pathway as the early checkpoint [14]. Ac-
ject to the regulation of environments. With regard to cordingly, the secretion of cellulase is a forward (antero-
the regulation mechanism at a genetic level, the type II grade) process rather than a backward (retrograde)
secretory pathway in E. carotovora subspecies carotovora process. In such a case, the secretory pathway should be
is encoded by the out cluster, which has 15 out genes regulated by Rab GTPase, which is a group of proteins
termed outB-0 and outs. This out cluster has been se- from the Ras superfamily of monomeric guanosine tri-
quenced and the resulting secretion defective mutants phosphatases and includes over 150 structurally closely
(Out-) have been isolated [115]. Thereislittle doubt that related members [13]. However, the Ras family is mainly
the list of genes involved in regulating the secretory found in humans. For example Rab27, an effector in the
pathway is increasing. For example Mdr49 is a func- regulation of secretory pathways, is not found in yeasts
tional homolog of Ste6 and mediates the ATP-binding and plants [127]. The consideration given to Rab27 is
cassette transporter in D. melanogaster [116]. With re- partially due to the fact that the cells that secrete cellu-
gard to the regulation mechanism at environmental lase are not professional secretory cells, while Rab27 is
level, Ca2+ plays a role in the secretory pathway [117]. involved in the secretion of exosomes in non-secretory
For instance, there is a Ca2+-dependent growth defect in- cells [128], which again supports the idea that the cells
duced by the PMR1-disrupted mutant [118-120]. There secreting cellulase are not specific secretory cells. On
are also many environmental factors that can affect the theonehand,endosomesdealwithincomingtraffic,that
functionalityofthesecretorypathway,suchaspH. is, to endocytose proteins, sort, recycle,and process deg-
radation of internalized cargos. However, on the other
Secretorymechanismsofcellulase hand, the exosomes in most cell types are derived from
A general consideration on the secretion of cellulases intraluminal vesicles of multivesicular endosomes, form
suggests that there are three different mechanisms based small membrane vesicles, and contribute to intercellular
on their subcellular locations: 1) a specific secretory communications [129].
pathwayindependent ofcellulose, 2)a secretory pathway Mutations can certainly change cellulase production,
which is induced by cellulose, and 3) a generalized bleb- forinstance,whengrownoncrystallinecellulose,asignifi-
bing process that occurs irrespective of the carbon cantlyhighamountofcellulasecanbesynthesizedandse-
source [36]. The cellulase secretion needs to be induced, creted by a mutant fungus of T. reesei QM6a (mutant
and this induction can include the generation of new RUT-C30).Onthecontrary,thecellulaseactivitydecreases
proteins for constructing secretory pathways. Taking an at latestagesof thegrowthof wild-type cells because they
example of induction of cellulases and hemicellulases by canonlysecreteasmallamountofcellulase[28].
D-xylose, Ferreira de Oliveira et al. found that 282 pro-
teins were induced by D-xylose and 161 proteins were SecretorymechanismdescribedinUniProtKB
induced by sorbitol, while another 638 proteins were Up until now, the literature review reveals that the
presented under both conditions in mycelia from A. secretory pathwayfor most cellulases is most likely to be
niger; of which 254 proteins were predicted to relate to thetypeIIsecretorypathway,forwhichthesignalregion
the secretory pathway [14]. The cellulose itself can also in cellulase plays a role to initiate the secretion of
YanandWuBiotechnologyforBiofuels2013,6:177 Page8of12
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cellulase into the extracellular matrix. Therefore, it is falciparum Ca2+-dependent protein kinase 1 in P. falcip-
notunnecessarytoexaminethesignalregionofcellulases arum,areexportedtotheparasitophorousvacuoleinpar-
in UniProtKB in order to gain a general overview of this asites [138]. These findings suggest that various lipidated
issue. Of 4,101 cellulases documented in UniProtKB, 386 peptides and proteins are secreted by ATP-binding cas-
cellulaseshavea signalregionintheirsequence.Withex- settetransportersineukaryoticorganisms[70].
ceptionof three cellulases, the lengthof signalpeptides is
27±7 (mean±SD) amino acids ranging from 16 to 65 Conclusions: implications and knowledge gaps
amino acids (Figure 2). It is said that the yeast secretary Although the secretory pathway is a popular research
signal is an alpha-factor fragment [130] and can be pre- topic, the present review indicates that this topic has yet
dicted using the PSORT program [131]. In UniProtKB to draw sufficient attention in cellulase research. How-
there are ten yeasts, whose signal regions are considered ever, the literature review suggests that cellulases share
tobesimilartothealpha-factorfragment. their secretory pathway with other secreted proteins ra-
However, 3,713 cellulases are yet to have the signal re- ther than having a specific secretory pathway. Therefore,
gion in their sequence annotated, which could be due to the knowledge obtained from studies on secretory path-
technical reasons or could otherwise suggest that the se- ways generally benefits our understanding on the
cretion of these cellulases go through other secretory secretory pathway ofcellulase.
pathways. This may be possible because the five types of Of the 4,101 cellulases in UniProtKB [5], a few were
secretory pathway, whose mechanisms are clearly illus- annotatedwith enzymatic activityofcellulasesunderdif-
trated, are referenced to Gram-negative bacteria. Indeed, ferent circumstances. For example the optimal pH for a
the mechanism that regulates the secretion of non- cellulase reaction is 5.26±1.4 (mean±SD, n=8) for the
vesicular proteins using the ATP-binding cassette trans- cellulases obtained from Eukaryota and 6.39±1.49
porter is important for biotechnology because this (mean±SD,n=11) for the cellulases obtained from bac-
pathway belongs to the type I pathway and seems to teria, while the optimal temperature for a cellulase reac-
translocate lipidated peptides and proteins across the tion is 53±11.58°C for the cellulases obtained from
plasma membrane of eukaryotic cells. For example the Eukaryotaand53.78±17.28°Cforthecellulasesobtained
yeast alpha-factor pheromone, a farnesylated peptide, is from bacteria.
transported in this manner by the ATP-binding cassette The literature review has revealed several gaps in
transporter Ste6[132,133], andthe farnesylatedM-factor knowledgewith regard tothesecretory pathwayofcellu-
of Schizosaccharomyces pombe is also transported by the lases,includingthefollowing:
ATP-bindingcassettetransporterMam1[134].Yet,acylated
proteins, such as the hydrophilic acylated surface protein 1) Thesubcellular locationofcellulasesis stillnotclear
B in Leishmania species [135-137] and Plasmodium insome organisms,suchasprotozoa,although some
studies havebeen undertaken[139].Atpresent,this
shortagecouldbecompensatedbythefactthat
protozoaareadiversegroupofunicellulareukaryotic
organisms[140].Therefore,ourunderstandingonthe
subcellularlocationsofcellulasesineukaryotescould
beappliedtoprotozoa.
2) Ascellulaseneedstobe coated withavesicle, itis
possiblethatcellulasescouldbeinvolved in
exosomesaswellasendosomes,whose function
deals with incoming traffic.Isitpossible that
cellulasescanbetransferred backinto cells,
consideringexperimental observations indicatea
declineinenzymatic activity withtime?
3) Itiswidely understood thatmany proteins needto
befolded inordertotransportthrough the
membrane(forexamplefibroblastgrowth factor 2
hastobefullyfolded topassthrough theplasma
membrane[141-143]). However,itisnotclear
whetheracellulaseneedstobefoldedinorderto
transportthrough thesecretorypathway.
Figure2Lengthdistributionofsignalpeptidesofcellulases
4) Thereisa shortageofstudies onthemechanisms
obtainedfromUniProtKBwiththeirsecretingorganisms.
regulatingthesecretory pathway ofcellulaseatboth
YanandWuBiotechnologyforBiofuels2013,6:177 Page9of12
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geneticand environmentallevels.Forinstance, itis Acknowledgements
notclearwhetherinhibitorscouldhelptostopthe ThisstudywaspartlysupportedbytheGuangxiScienceFoundation
(12237022,1347004–1,2013GXNSDA019007,13-051-08,and13-051-50)and
secretionofother proteins that sharethesame
BaGuiScholarsProgramFoundation.
secretory pathway with cellulase.Itisalsonotclear
whetherosmolalityinfluencesthesecretorypathway Received:2October2013Accepted:19November2013
Published:2December2013
ofcellulase,sinceit wouldbeexpectedthatthe
concentrations ofsubstrateand product change
continuously duringfermentationleading the References
osmolalitytochangetogetherwithpH, temperature, 1. Cellulase:TheAmericanHeritageScienceDictionary.Dictionary.com.Boston,MA:
HoughtonMifflinCompany.http://dictionary.reference.com/browse/cellulase.
pressure,and soon.
2. Cellulose:Dictionary.comUnabridged.Dictionary.com.NewYork,NY:Random
5) Aconsiderablenumber ofstudies havebeen House,Inc.http://dictionary.reference.com/browse/cellulose.
undertaken tomutate genesinordertoenhancethe 3. EC3.2.1.4–cellulase:BRENDA.Brunswick:InstituteofBiochemistryand
BioinformaticsattheTechnicalUniversityofBrunswick.http://www.brenda-
activity ofcellulase[144,145].However,the
enzymes.org/php/result_flat.php4?ecno=3.2.1.4.
literaturereviewcouldsuggestawaytoincreasethe 4. WatanabeH,TokudaG:Cellulolyticsystemsininsects.AnnuRevEntomol
secretionofcellulasethroughthe mutations inthe 2010,55:609–632.
5. ConsortiumUP:TheUniversalProteinEesource(UniProt)in2010.Nucleic
secretory pathway ofcellulase.Forexample a AcidsRes2010,38:D142–D148.
mutationinthePMR1gene resultedina5-to 6. ParawiraW:Enzymeresearchandapplicationsinbiotechnological
50-foldincreaseinthesecretionof bovinegrowth intensificationofbiogasproduction.CritRevBiotechnol2012,32:172–186.
7. WatanabeH,TokudaG:Animalcellulases.CellMolLifeSci2001,58:1167–1178.
hormone,prochymosin [121,146].
8. CoughlanMP:Thepropertiesoffungalandbacterialcellulaseswith
6) Asmost secretoryand cell-surfaceproteinscontain commentontheirproductionandapplication.BiotechnolGenetEngRev
disulfidebonds[147,148],it isnotclearwhethercel- 1985,3:39–109.
9. CoughlanMP,LjungdahlLG:Comparativebiochemistryoffungaland
lulasesshouldundergothe processtohavedisulfide
bacterialcellulolyticsystems.FEMSSymp1988,43:11–30.
bonds duringtransportation, whilethe cellulases 10. FinkelsteinDB:ImprovementofenzymeproductioninAspergillus.Antonie
thatdo havedisulfidebonds areamongthe4,101 VanLeeuwenhoek1987,53:349–352.
11. HeslotH:Geneticsandgeneticengineeringoftheindustrialyeast
cellulasesfrom UniProtKB,suchasthe cellulase
Yarrowialipolytica.AdvBiochemEngBiotechnol1990,43:43–73.
[UniProt:P07103]. 12. DashtbanM,SchraftH,QinW:Fungalbioconversionoflignocellulosic
residues;opportunities&perspectives.IntJBiolSci2009,5:578–595.
13. HilbiH,HaasA:Secretivebacterialpathogensandthesecretorypathway.
Abbreviations
Traffic2012,13:1187–1197.
CMCase:Carboxymethylcellulase;COPII:CoatproteincomplexII;
14. deOliveiraJMF,vanPasselMW,SchaapPJ,deGraaffLH:Shotgun
ERGIC:Endoplasmicreticulum-to-Golgiintermediatecompartment;FEA:Flagella
proteomicsofAspergillusnigermicrosomesuponD-Xyloseinduction.
exportapparatus;FPE:Fimbrilin-proteinexporter;GSP:Generalsecretory
ApplEnvironMicrobiol2010,76:4421–4429.
pathway;holin:Holeforming;Sec:Secretion;Tat:Twin-argininetranslocation;
15. TolmachovaT,AndersR,StinchcombeJ,BossiG,GriffithsGM,HuxleyC,
UniProtKB:UniProtKnowledgebase;Wss:WXG100secretionsystem.
SeabraMC:AgeneralroleforRab27ainsecretorycells.MolBiolCell2004,
15:332–344.
Competinginterests 16. FukudaM:VersatileroleofRab27inmembranetrafficking:focusonthe
Theauthorsdeclarethattheyhavenocompetinginterests. Rab27effectorfamilies.JBiochem2005,137:9–1316.
17. FukudaM:RegulationofsecretoryvesicletrafficbyRabsmallGTPases.
CellMolLifeSci2008,65:2801–2813.
Authors’contributions
18. BoothAE,SeabraMC,HumeAN:Rab27aandmelanosomes:amodelto
GWconceivedtheideaandwrotethefirstdraftofthemanuscript.SMYand
investigatethemembranetargetingofRabs.BiochemSocTrans2012,
GWcontributedtomanuscriptrevisionandapprovedthefinalversion. 40:1383–1388.
19. ChouKC,ShenHB:Euk-mPLoc:afusionclassifierforlarge-scale
Authors’information eukaryoticproteinsubcellularlocationpredictionbyincorporating
GWreceivedhisMDfromTianjinMedicalUniversity,Tianjin,China(1984), multiplesites.JProteomeRes2007,6:1728–1734.
PhDfromRussianMedicalUniversity,Moscow,Russia(1992),did 20. ChouKC,ShenHB:Cell-PLoc:ApackageofWebserversforpredicting
postdoctoralresearchatUniversityofUdine,Udine,Italy(1992to1993), subcellularlocalizationofproteinsinvariousorganisms.NatProtoc2008,
InstituteforMinamataDisease,Minamata,Japan(1993to1994),Universityof 3:153–162.
Udine,Udine,Italy(1994to1999),UniversityofMarseilles,Marseilles,France 21. MaronBA,MichelT:Subcellularlocalizationofoxidantsandredox
(2000),andworkedatNovartisPharmaAG,Basel,Switzerland(2001to2002). modulationofendothelialnitricoxidesynthase.CircJ2012,76:2497–2512.
GWworkedasageneralmanageratDreamSciTechConsultingcompany, 22. DickJM:Calculationoftherelativemetastabilitiesofproteinsin
Shenzhen,China(2002to2012),andsince2008,hasworkedasaguest subcellularcompartmentsofSaccharomycescerevisia.BMCSystBiol2009,
professoratGuangxiAcademyofSciences,Nanning,China.GWhas 3:75.
publishedover170researchpapers,includingover120researchpapersin 23. BalAK,VermaDPS,ByrneH,MacLachlanGA:Subcellularlocalizationof
ScienceCitationIndex(SCI)journalsandsixbooksintheUSAandGermany. cellulasesinauxin-treatedpea.JCellBiol1976,69:97–105.
SMYreceivedherMDfromTianjinMedicalUniversity,Tianjin,China(1984), 24. DesvauxM,ParhamMJ,HendersonIR:TypeVproteinsecretion:simplicity
MSfromTianjinMedicalUniversity,Tianjin,China(1987),PhDfromUniversity goneawry.CurrIuuesMolBiol2004,6:111–124.
ofSiena,Siena,Italy(2000),anddidpostdoctoralresearchatUniversityof 25. HillTW,MullinsJT:HyphaltipgrowthinAchlya:II.subcellularlocalization
Udine,Udine,Italy(1999to2001).SMYworkedasadirectoratDreamSciTech ofcellulaseandassociatedenzymes.CanJMicrobiol1980,26:1141–1146.
Consultingcompany,Shenzhen,China(2002to2012),andsince2008,has 26. GhoshA,Al-RabiaiS,GhoshBK,Trimino-VasquezH,EveleighDE,Montenecourt
beenworkingasaprofessoratGuangxiAcademyofSciences,Nanning, BS:IncreasedendoplasmicreticulumcontentofamutantofTrichloderma
China.SMYhaspublishedover140researchpapersincludingover80 reesei(Rut-C30)inrelationtocellulasesynthesis.EnzymeMicrobTechnol
researchpapersinSCIjournalsandtwobooksintheUSAandGermany. 1982,4:110–113.
YanandWuBiotechnologyforBiofuels2013,6:177 Page10of12
http://www.biotechnologyforbiofuels.com/content/6/1/177
27. GhoshA,GhoshBK,Trimino-VazquezH,EveleighDE,MontenecourtBS: 52. BlockerA,KomoriyaK,AizawaS:TypeIIIsecretionsystemsandbacterial
CellulasesecretionfromahypercellulolyticmutantofTrichoderinareesei flagella,insightsintotheirfunctionfromstructuralsimilarities.ProcNatl
Rut-C30.ArchMicrobiol1984,140:126–133. AcadSciUSA2003,100:3027–3030.
28. GlennM,GhoshA,GhoshBK:Subcellularfractionationofa 53. GauthierA,ThomasNA,FinlayBB:Bacterialinjectionmachines.JBiol
hypercellulolyticmutant,TrichodermareeseiRut-C30:localizationof Chem2003,278:25273–25276.
endoglucanaseinmicrosomalfraction.ApplEnvironMicrobiol1985, 54. ChristiePJ,VogelJP:BacterialtypeIVsecretion,conjugationsystemsadapted
50:1137–1143. todelivereffectormoleculestohostcells.TrendsMicrobiol2000,8:354–360.
29. CaiYJ,ChapmanSJ,BuswellJA,ChangST:Productionanddistributionof 55. ChristiePJ:TypeIVsecretion,intercellulartransferofmacromoleculesby
endoglucanase,cellobiohydrolase,andbeta-glucosidasecomponentsof systemsancestrallyrelatedtoconjugationmachines.MolMicrobiol2001,
thecellulolyticsystemofVolvariellavolvacea,theediblestraw 40:294–305.
mushroom.ApplEnvironMicrobiol1999,65:553–559. 56. YenMR,PeabodyCR,PartoviSM,ZhaiY,TsengYH,SaierMH:Protein-
30. GardnerRG,WellsJE,RussellJB,WilsonDB:ThecellularlocationofPrevotella translocatingoutermembraneporinsofgram-negativebacteria.Biochim
ruminicolabeta-1,4-D-endoglucanaseanditsoccurrenceinotherstrainsof BiophysActa2002,1562:6–31.
ruminalbacteria.ApplEnvironMicrobiol1995,61:3288–3292. 57. MougousJD,CuffME,RaunserS,ShenA,ZhouM,GiffordCA,GoodmanAL,
31. RussellJB:Fermentationofcellodextrinsbycellulolyticand JoachimiakG,OrdonězCL,LoryS,WalzT,JoachimiakA,MekalanosJJ:A
noncellulolyticrumenbacteria.ApplEnvironMicrobiol1985,49:572–576. virulencelocusofPseudomonasaeruginosaencodesaproteinsecretion
32. MatsushitaO,RussellJB,WilsonDB:Cloningandsequencingofa apparatus.Science2006,312:1526–1530.
BacteroidesruminicolaB14endoglucanasegene.JBacteriol1990, 58. PukatzkiS,MaAT,SturtevantD,KrastinsB,SarracinoD,NelsonWC,
172:3620–3630. HeidelbergJF,MekalanosJJ:Identificationofaconservedbacterial
33. AvgustinG,FlintHJ,WhiteheadTR:Distributionofxylanasegenesand proteinsecretionsysteminVibriocholeraeusingtheDictyosteliumhost
enzymesamongstrainsofPrevotella(Bacteroides)ruminicolafromthe modelsystem.ProcNatlAcadSciUSA2006,103:1528–1533.
rumen.FEMSMicrobiolLett1992,99:137–144. 59. BoyerF,FichantG,BerthodJ,VandenbrouckY,AttreeI:Dissectingthe
34. VercoePE,GreggK:DNAsequenceandtranscriptionofan bacterialtypeVIsecretionsystembyagenomewideinsilicoanalysis:
endoglucanasegenefromPrevotella(Bacteroides)ruminicolaAR20. whatcanbelearnedfromavailablemicrobialgenomicresources?BMC
MolGenGenet1992,233:284–292. Genomics2009,10:104.
35. WhiteheadTR:Analysesofthegeneandaminoacidsequenceofthe 60. BingleLE,BaileyCM,PallenMJ:TypeVIsecretion:abeginner’sguide.Curr
Prevotella(Bacteroides)ruminicola23xylanaserevealsunexpected OpinMicrobiol2008,11:3–8.
homologywithendoglucanasesfromothergeneraofbacteria. 61. FillouxA,HachaniA,BlevesS:ThebacterialtypeVIsecretionmachine:yet
CurrMicrobiol1993,27:27–33. anotherplayerforproteintransportacrossmembranes.Microbiology
36. McGavinM,LamJ,ForsbergCW:Regulationanddistributionof 2008,154:1570–1583.
Fibrobactersuccinogenessubsp.succinogenesS85endoglucanases. 62. ChampionPA,ChampionMM,ManzanilloP,CoxJS:ESX-1secreted
ApplEnvironMicrobiol1990,56:1235–1244. virulencefactorsarerecognizedbymultiplecytosolicAAAATPasesin
37. GroleauD,ForsbergCW:Thecellulolyticactivityoftherumenbacterium pathogenicmycobacteria.MolMicrobiol2009,73:950–962.
Bacteroidessuccinogenes.CanJMicrobiol1981,27:517–530. 63. BitterW,HoubenEN,BottaiD,BrodinP,BrownEJ,CoxJS,DerbyshireK,
38. GroleauD,ForsbergCW:Partialcharacterizationoftheextracellular FortuneSM,GaoLY,LiuJ,vanPittiusNCG,PymAS,RubinEJ,ShermanDR,
endoglucanaseproducedbyBacteroidessuccinogenes.CanJMicrobiol ColeST,BroschR:SystematicgeneticnomenclaturefortypeVIIsecretion
1983,29:504–517. systems.PLoSPathog2009,10:e1000507.
39. SchelHhornHE,ForsbergCW:Multiplicityofextracellular1-(1,4)-endoglucanases 64. DalekeMH,UmmelsR,BawonoP,HeringaJ,Vandenbroucke-GraulsCM,
ofBacteroidessuccinogenesS85.CanJMicrobiol1984,30:930–937. LuirinkJ,BitterW:GeneralsecretionsignalforthemycobacterialtypeVII
40. McGavinM,ForsbergCW:Isolationandcharacterizationof secretionpathway.ProcNatlAcadSciUSA2012,109:11342–11347.
endoglucanases1and2fromBacteroidessuccinogenes.JBacteriol1988, 65. DesvauxM,HébraudM,TalonR,HendersonIR:Secretionandsubcellular
170:2914–2922. localizationsofbacterialproteins:asemanticawarenessissue.Trends
41. McGavinMJ,ForsbergCW,CrosbyB,BellAW,DignardD,ThomasDY: Microbiol2009,17:139–145.
Structureofthecel-3genefromFibrobactersuccinogenesS85and 66. RoschJ,CaparonM:Amicrodomainforproteinsecretioningram-
characteristicsoftheencodedgeneproduct,endoglucanase3.JBacteriol positivebacteria.Science2004,304:1513–1515.
1989,171:5587–5595. 67. DiaoL,DongQ,XuZ,YangS,ZhouJ,FreudlR:Functionalimplementation
42. HuangL,ForsbergCW,ThomasDY:Purificationandcharacterizationofa oftheposttranslationalSecB-SecAprotein-targetingpathwayinBacillus
chloride-stimulatedcellobiosidasefromBacteroidessuccinogenes. subtilis.ApplEnvironMicrobiol2012,78:651–659.
JBacteriol1988,170:2923–2932. 68. AndersonM,ChenYH,ButlerEK,MissiakasDM:EsaD,asecretionfactorfor
43. HuangL,ForsbergCW:IsolationofacellodextrinasefromBacteroides theEsspathwayinStaphylococcusaureus.JBacteriol2011,193:1583–1589.
succinogenes.ApplEnvironMicrobiol1987,53:1034–1041. 69. FagerlundA,LindbäckT,GranumPE:BacilluscereuscytotoxinsHbl,Nhe
44. HuangL,ForsbergCW:Cellulosedigestionandcellulaseregulationand andCytKaresecretedviatheSectranslocationpathway.BMCMicrobiol
distributioninFibrobactersuccinogenessubsp.succinogenesS85. 2010,10:304.
ApplEnvironMicrobiol1990,56:1221–1228. 70. RabouilleC,MalhotraV,NickelW:Diversityinunconventionalprotein
45. SalmondGP,ReevesPJ:Membranetrafficwardensandproteinsecretion secretion.JCellSci2012,125:5251–5255.
ingram-negativebacteria.TrendsBiochemSci1993,18:7–12. 71. EngelmanDM,SteitzTA:Thespontaneousinsertionofproteinsintoand
46. HendersonIR,NataroJP,KaperJB,MeyerTF,FarrandSK,BurnsDL,FinlayBB, acrossmembranes:thehelicalhairpinhypothesis.Cell1981,23:411–422.
StGemeJW3rd:Renamingproteinsecretioninthegram-negative 72. HendersonR,UnwinPNT:Three-dimensionalmodelofpurplemembrane
bacteria.TrendsMicrobiol2000,8:352. obtainedbyelectronmicroscopy.Nature1975,257:28–32.
47. ThanassiDG,HultgrenSJ:Multiplepathwaysallowproteinsecretion 73. OzolsJ,GerardC:Covalentstructureofthemembranoussegmentof
acrossthebacterialoutermembrane.CurrOpinCellBiol2000,12:420–430. horsecytochromeb5.JBiolChem1977,252:8549–8553.
48. AndersenC:Channel-tunnels,outermembranecomponentsoftypeI 74. SpangA:OnvesicleformationandtetheringintheER-Golgishuttle.
secretionsystemsandmultidrugeffluxpumpsofgram-negative CurrOpinCellBiol2009,21:531–536.
bacteria.RevPhysiolBiochemPharmacol2003,147:122–165. 75. MatobaS,FukayamaJ,WingRA,OgrydziakDM:Intracellularprecursors
49. StathopoulosC,HendrixsonDR,ThanassiDG,HultgrenSJ,StGemeJW3rd, andsecretionofalkalineextracellularproteaseofYarrowialipolytica.
CurtissR3rd:Secretionofvirulencedeterminantsbythegeneral MolCellBiol1988,8:4904–4916.
secretorypathwayingram-negativepathogens,anevolvingstory. 76. NicaudJM,FournierP,BonnardiereCL,ChaslesM,GaillardinC:Useofars18
MicrobesInfect2000,2:1061–1072. basedvectorstoincreaseproteinproductioninYarrowialipolytica.
50. SandkvistM:BiologyoftypeIIsecretion.MolMicrobiol2001,40:271–283. JBiotechnol1991,19:259–270.
51. ButtnerD,BonasU:Portofentry,thetypeIIIsecretiontranslocon.Trends 77. DelicM,ValliM,GrafAB,PfefferM,MattanovichD,GasserB:Thesecretory
Microbiol2002,10:186–192. pathway:exploringyeastdiversity.FEMSMicrobiolRev2013,37:872–914.