Báo cáo khoa học: The mystery of nonclassical protein secretion A current view on cargo proteins and potential export routes pdf

REVIEW ARTICLE
The mystery of nonclassical protein secretion
A current view on cargo proteins and potential export routes
Walter Nickel
Biochemie-Zentrum Heidelberg, University of Heidelberg, Germany
Most of the examples of protein translocation across a
membrane (such as the import of classical secretory proteins
into the endoplasmic reticulum, import of proteins into
mitochondria and peroxisomes, as well as protein import
into and export from the nucleus), are understood in great
detail. In striking contrast, the phenomenon of unconven-
tional protein secretion (also known as nonclassical protein
export or ER/Golgi-independent protein secretion) from
eukaryotic cells was discovered more than 10 years ago and
yet the molecular mechanism and the molecular identity of
machinery components that mediate this process remain
elusive. This problem appears to be even more complex as
several lines of evidence indicate that various kinds of
mechanistically distinct nonclassical export routes may exist.
In most cases these secretory mechanisms are gated in a
tightly controlled fashion. This review aims to provide a
comprehensive overview of our current knowledge as a basis
for the development of new experimental strategies designed
to unravel the molecular machineries mediating ER/Golgi-
independent protein secretion. Beyond solving a funda-
mental problem in current cell biology, the molecular
analysis of these processes is of major biomedical importance
as these export routes are taken by proteins such as angio-
genic growth factors, inflammatory cytokines, components
of the extracellular matrix which regulate cell differentiation,
proliferation and apoptosis, viral proteins, and parasite

surface proteins potentially involved in host infection.
Keywords: unconventional protein secretion; nonclassical
export; protein targeting; membrane translocation; extra-
cellular localization; FGF-2 trafficking; galectin trafficking;
Leishmania HASPB trafficking; interleukin 1a and 1b
trafficking; ER/Golgi-independent protein secretion.
Introduction
Soluble secretory proteins typically contain N-terminal
signal peptides that direct them to the translocation
apparatus of the endoplasmic reticulum (ER) [1]. Following
vesicular transport from the ER via the Golgi to the cell
surface, lumenal proteins are released into the extracellular
space by fusion of Golgi-derived secretory vesicles with the
plasma membrane [2–5]. This pathway of protein export
from eukaryotic cells is known as the classical or ER/Golgi-
dependent secretory pathway. However, more than 10 years
ago, it was reported that interleukin 1b (IL1b) and galectin-1
(alsoreferredtoasL-14)couldbeexportedfromcellsinthe
absence of a functional ER/Golgi system [6,7]. Since then,
the list of proteins demonstrated to be secreted by uncon-
ventional means is steadily growing. Figure 1 gives an
overview of cellular, viral and parasitic proteins that have
been shown to be exported by mechanisms that are
operational in the absence of a functional ER/Golgi system.
The basic observations (summarized previously in [8,9]) that
led to the proposal of alternative pathways of eukaryotic
protein secretion are (a) the lack of conventional signal
peptides in the secretory proteins in question, (b) the
exclusion of these proteins from classical secretory organel-
les such as the ER and the Golgi combined with the lack of

ER/Golgi-dependent post-translational modifications such
as N-glycosylation and (c) resistance of these export
processes to brefeldin A, a classical inhibitor of ER/Golgi-
dependent protein secretion [10–12]. Because the secretory
proteins discussed here are soluble factors synthesized on
free ribosomes in the cytoplasm, various experimental
strategies have been pursued in order to exclude unspecific
release based on cell death under the experimental condi-
tions applied. As described in detail in the following
sections, these experiments included parallel quantitative
measurements of the appearance of unrelated cytoplasmic
proteins in cellular supernatants [8,9] as well as the
identification of mutants that are deficient in nonclassical
export [13]. Moreover, nonconventional protein secretion
was shown to be dependent on both energy and temperature
and is stimulated or inhibited by various treatments [8,9].
Finally, nonconventional protein secretion processes were
shown to be regulated for example by cell differentiation
[7,14], NF-jB-dependent signalling pathways [15], and post-
translational modifications such as phosphorylation [16].
Based on these observations, it has to be concluded that the
secretory proteins discussed in this review exit eukaryotic
cells in a controlled manner mediated by proteinaceous
machineries. In the following sections, the various cargo
proteins known to be secreted by unconventional means will
be discussed in detail.
Correspondence to W. Nickel, Biochemie-Zentrum Heidelberg,
University of Heidelberg, Im Neuenheimer Feld 328,
69120 Heidelberg, Germany.
E-mail:

Abbreviations: ER, endoplasmic reticulum; FGF, fibroblast
growth factor.
(Received 12 February 2003, accepted 17 March 2003)
Eur. J. Biochem. 270, 2109–2119 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03577.x
Cytokines: interleukin-1b, thioredoxin
and macrophage migration inhibitory factor
In 1987, Dinarello and colleagues demonstrated that
interleukin 1, a cytokine [17,18] lacking a classical signal
peptide for ER/Golgi-mediated protein secretion [19], is
exported from activated human monocytes [20]. Two
isoforms of interleukin 1 termed IL-1a and IL-1b have
been described which represent proteolytically processed
forms derived from two related but distinct precursors [18].
The processing of IL-1a involves myristoylation and,
following insertion into the plasma membrane, calpain-
dependent cleavage that is thought to cause release of the
mature form of IL-1a into the extracellular space [21,22]. In
the case of IL-1b, interleukin-converting enzyme produces
mature IL-1b [23,24], which is then exported [18].
Most studies targeted toward the molecular mechanism
of interleukin 1 export have been focused on the b-isoform.
A detailed molecular analysis of the export process revealed
that IL-1b does not make use of an unconventional
pathway of translocation into the lumen of the endoplasmic
reticulum but rather appears to utilize a secretory mechan-
ism independent of ER/Golgi-related vesicular transport [6].
This process was shown to be distinct from unspecific
release as for example only the processed form of IL-1b
(17 kDa) can be detected in cellular supernatants whereas
the precursor (33 kDa) is retained by IL-1b-expressing cells

[6]. Moreover, under the experimental conditions applied,
only the b-isoform was found to be secreted, whereas the
a-isoform could not be detected in cellular supernatants
[6]. However, despite apparently utilizing a distinct secretory
mechanism, it was later found that IL-1a is also exported
[21].
Though IL-1b is found in certain intracellular vesicles, as
judged by protease protection experiments, these structures
appear to be unrelated to the ER/Golgi system as IL-1b
secretion was not inhibited but rather stimulated by
brefeldin A, a drug that compromises the structure and
function of the Golgi apparatus [10–12]. Consistently, IL-1b
was found not to be glycosylated, despite bearing corres-
ponding consensus sequences. Intracellular vesicles pro-
posed to play a role in IL-1b secretion have been shown to
be related to an endolysosomal compartment that releases
its content upon fusion with the plasma membrane [25].
These observations are consistent with the fact that IL-1b
secretion is sensitive to methylamine [6], a drug that disturbs
endocytosis [26]. Based on pharmacological studies
employing the sulfonylurea glyburide (10 l
M
) along with
expression-inhibition studies employing antisense tech-
niques, an ABC transporter, ABC1, has been implicated
in the overall process of IL-1b secretion [27,28] and
therefore might mediate IL-1b translocation from the
cytoplasm to the lumen of the endolysosomal compartment.
Interestingly, glyburide also appears to inhibit nonclassi-
cal secretion of macrophage migration inhibitory factor

(J. Bernhagen, RWTH Aachen, Germany, personal com-
munication), an inflammatory cytokine mediating a number
of immune and inflammatory diseases, e.g. bacterial septic
shock [29–31]. The potential function of ABC transporters
in these processes might be related to that of bacterial ABC
transporters that mediate protein secretion of, for example,
hemolysin [32–34].
Fig. 1. Cargo proteins and potential export
routes of unconventional protein secretion. At
least four distinct types of nonclassical export
can be distinguished. For IL-1b,En2and
HMGB1, export involves import into intra-
cellular vesicles, which are probably endo-
somal subcompartments. FGF-1 and FGF-2
probably reach the extracellular space by
direct translocation across the plasma mem-
brane, but they apparently use distinct trans-
port systems. The Leishmania cell surface
protein HASPB also translocates directly
across the plasma membrane and requires that
the protein is membrane-anchored through
dual acylation at the N-terminus. Therefore, a
flip-flop mechanism is required to locate the
protein in the outer leaflet of the plasma
membrane. The final postulated pathway of
unconventional protein secretion involves the
formation of exosomes, vesicles that form on
the outer surface of the cell in a process known
as membrane blebbing. Exosomes are labile
structures that release their contents into the

extracellular space. It has been suggested that
this pathway may be used by the galectins.
2110 W. Nickel (Eur. J. Biochem. 270) Ó FEBS 2003
Thioredoxins are ubiquitous intracellular enzymes that
catalyze thiol-disulfide exchange reactions [35]. Additionally,
extracellular populations of thioredoxin have been detected
that, similar to IL-1b and migration inhibiting factor, follow
an ER/Golgi-independent route of secretion [36–39]. This
observation is consistent with additional physiological roles
of thioredoxin such as its function as a mitogenic cytokine
that requires extracellular localization [40,41]. Secretion of
thioredoxin appears to be mediated by a pathway distinct
from IL-1b as it could neither be detected in intracellular
vesicles, nor was the secretion process reported to be
inhibited by reagents that interfere with the function of
ABC transporters. However, as with IL-1b [6], secretion of
thioredoxin is inhibited by methylamine and stimulated by
brefeldin A [39]. Interestingly, the redox state of thioredoxin
does not influence its unconventional export [42].
Pro-angiogenic growth factors:
FGF-1 and FGF-2
Fibroblast growth factor 1and 2 (FGF-1 and FGF-2) belong
to a large family of heparin-binding growth factors [43] that,
apart from their mitogenic activity [43,44], are key activators
of tumor-induced angiogenesis [45]. The majority of the
members of the FGF family are exported by ER/Golgi-
dependent secretory transport. However, FGF-1 and the
18 kDa isoform of FGF-2 have been shown to be secreted by
an alternative pathway [46–48]. While it was first assumed
that angiogenic growth factors might be released from

mechanically injured tissue to promote wound healing [49], a
process that requires angiogenesis, various lines of evidence
demonstrate that FGF-1 and FGF-2 are exported from
cultured cells in the absence of appreciable amounts of cell
death [46–48,50,51]. Like IL-1b [6], FGF-1 is increasingly
secreted under stress conditions such as heat shock treatment
[46,52]. In contrast, FGF-2 export is not affected under these
experimental conditions [53]. While serum starvation has
been reported to inhibit export of both FGF-2 [48] and IL-1b
[6], it was found to actually induce secretion of FGF-1 [52].
Similarly, methylamine has been found to block export only
of FGF-2 [48] and IL-1b [6] with no apparent effect on FGF-
1 export [54]. Recently, it was reported that expression of the
IL-1a precursor inhibits FGF-1 release in response to
temperature stress [55]. In contrast, expression of the mature
form of IL-1a did not affect FGF-1 export, suggesting that
IL-1a processing is somehow related to FGF-1 biogenesis.
However, whether FGF-1 and IL-1a utilize similar export
mechanisms remains an open question.
These observations point to some common characteristics
in the export of the cargo proteins discussed, but it seems
unlikely that one and the same machinery mediates secretion
of these factors. Consistent with this view, IL-1b has been
reported to be secreted by a vesicular nonclassical export
pathway [6,25], while FGF-1 and FGF-2 are likely to be
directly translocated from the cytoplasm into the extracel-
lular space (Fig. 1). While there is also one report pointing to
a role of large granules involved in FGF-2 export based on
immuno-EM analysis of mast cells [56], this issue remains
controversial as intracellular FGF-2 has been localized to the

cytoplasm in many FGF-2-secreting cell types with no
apparent localization in vesicular structures [50,57–59].
Similar findings have been reported for FGF-1 [60–62].
With regard to the protein components involved in the
overall processes of nonclassical export pathways, most is
known about the secretion of FGF-1. As noted above,
FGF-1 export is significantly increased in response to stress
conditions such as heat shock treatment [46] and serum
starvation [52]. Based on these experimental conditions, it
was shown that secreted FGF-1 isolated from cell culture
supernatants represents a latent (inactive form that can be
reactivated) homodimer [54] that can also be formed upon
chemical oxidation of FGF-1 in vitro [63]. These observa-
tions led to the discovery of a specific cysteine residue
(Cys30) in FGF-1 that is required for both dimer formation
and nonclassical export of FGF-1 [13,54]. Upon heat shock
treatment, two intracellular proteins have been shown to
associate with the latent FGF-1 homodimer in the cyto-
plasm. These are a cleavage product of the transmembrane
protein synaptotagmin consisting of its cytoplasmic domain
(p40-Syt1) and the Ca
2+
-binding protein S100A13. Appar-
ently, they are exported together with FGF-1 [64–66]. A
direct role of p40-Syt1 and S100A13 in FGF-1 export has
been proposed as both repression of p40-Syt1 expression by
antisense techniques and the expression of a dominant-
negative S100A13 mutant attenuate FGF-1 export [64,66].
As with FGF-1 dimer formation [63], oxidation by Cu
2+

cations has been demonstrated to trigger the formation of a
complex consisting of FGF-1, p40-Syt1 and S100A13 [67].
Consistent with the view that p40-Syt1 and S100A13 are
involved in the export of FGF-1, tetrathiomolybdate, a
Cu
2+
chelator, has been shown to inhibit heat shock-
induced FGF-1 export [67]. More recently, stress-induced
formation of the intracellular FGF-1–p40-Syt1–S100A13
complex has been demonstrated to cause a redistribution of
cytoplasmic FGF-1 to the inner surface of the plasma
membrane [62]. These results suggest that FGF-1–p40-
Syt1–S100A13 complex formation is the first step in the
FGF-1 export pathway, followed by direct translocation of
this protein complex across the plasma membrane. How-
ever, the machinery that mediates membrane translocation
of this protein complex remains unknown.
Compared to FGF-1 export, much less is known about
the mechanism and the role of specific proteins with regard
to the overall process of FGF-2 export from mammalian
cells. To date the only protein that has been proposed to
play a role in FGF-2 export is the Na
+
/K
+
-ATPase [68].
This conclusion was based initially on the observation that
cardiac glycosides such as ouabain partially inhibit FGF-2
export [50,68,69]. This was further strengthened by experi-
ments demonstrating that the expression of an ouabain-

resistent a-subunit mutant of the Na
+
/K
+
-ATPase rescues
FGF-2 export in the presence of ouabain [70]. Moreover, a
direct or indirect physical interaction between the a subunit
and FGF-2 has been detected based on coimmunopreci-
pitation though this association could only be observed
upon co-overexpression of both proteins [68]. Together with
the result that overexpression of the a subunit interferes with
FGF-2 export [68], these observations are reasonably
supportive of a role for the Na-K-ATPase in the overall
process of FGF-2 export. On the other hand, ouabain
treatment (typically used at 10–100 l
M
) causes only partial
inhibition of FGF-2 export, whereas concentrations of
ouabain of less than 5 l
M
(IC
50
 1 l
M
) completely inhibit
the ATP-dependent translocation of cations catalyzed by
Ó FEBS 2003 Nonclassical protein secretion (Eur. J. Biochem. 270) 2111
the Na
+
/K

+
-ATPase [71,72]. Interestingly, the membrane
potential generated by the Na
+
/K
+
-ATPase is not required
for FGF-2 export [68]. Based on these observations, it has
been proposed that the a/b heterodimers that constitute a
functional Na
+
/K
+
-ATPase in terms of ion transport
might be able to form higher ordered complexes that
catalyze FGF-2 export in a membrane potential-independ-
ent manner [68]. Alternatively, the a subunit alone might
associate with other so far uncharacterized factors as part of
a novel complex that mediates FGF-2 export [68]. Unfor-
tunately, no progress has yet been made in identifying such
molecular structures.
Galectins: components of the extracellular
matrix
The members of the galectin protein family are abundant
b-galactoside-specific lectins of the extracellular matrix
implicated in many cellular processes such as regulation of
cell proliferation, differentiation and apoptosis [73–76]. The
best characterized members of this family are galectin-1 and
galectin-3 which are present as soluble proteins in the
cytoplasm in a wide range of vertebrate cell lines and tissues

[7,14,77–81]. Secreted galectins are found either bound to the
extracellular surface of the plasma membrane or as abundant
components of the extracellular matrix [7,14,77,79–81]. Cell
surface association of galectins is mediated by both N- and
O-glycosylated b-galactose-terminated oligosaccharide side
chains of glycoproteins [9,73] as well as by galactose-
containing glycolipids such as GM
1
[73,82]. As galectin-1
and galectin-3 can form homodimers [9,83,84], it has been
proposed that secreted galectins affect their glycosylated cell-
surface counter receptors by inducing conformational chan-
ges of their extracellular domains and/or by clustering
galectin counter receptors based on noncovalent crosslinking
of oligosaccharide moieties [73]. In this way, secreted
galectins are thought to affect processes such as cell
differentiation by cell surface counter receptor-mediated
signalling [73,85]. While classical counter receptors of, for
example, galectin-1 include laminin [86], fibronectin [87] and
cell-type specific receptors such as T cell CD43 and CD45
[75], it has been shown more recently that the tumor-specific
cell surface antigen CA125 also represents a galectin counter
receptor that preferentially binds galectin-1 [79]. This latter
example is of particular interest as it provides a potential
molecular mechanism for how tumor cells can differentially
interact with the extracellular matrix, a process crucial for
tumor progression.
Similar to interleukin 1b, FGF-1 and FGF-2, galectins
apparently do not contain signal peptides in their primary
structure suitable for ER/Golgi-mediated secretion [88].

Consistently, galectins are synthesized on free ribosomes in
the cytoplasm [89] and galectin secretion has been shown
not to be blocked by inhibitors of the ER/Golgi-dependent
pathway such as brefeldin A and monensin [9,80,90]. Unlike
interleukin 1b, galectin-1 and galectin-3 do not appear to be
packaged into intracellular vesicles prior to export
[7,9,80,81]. Rather, galectin-1 and galectin-3 have been
shown to accumulate directly below the plasma membrane,
followed by an export mechanism that appears to involve
the formation of membrane-bound vesicles (also called
exosomes but not to be confused with structures involved in
RNA processing [91]) that pinch off before being released
into the extracellular space [7,9,80,81]. This mechanism also
distinguishes galectin export from FGF-1 and FGF-2
export, as there is no evidence that these proteins are
packaged into exosomes (see above). An engineered version
of galectin-3 containing an N-terminal acylation motif
derived from a protein tyrosine kinase (p56
lck
) has been
showntobesecretedmoreefficientlythanwild-type
galectin-3 [81]. These results indicate that targeting to the
plasma membrane is a rate-limiting step in galectin secre-
tion. However, there is no information describing exactly
what causes galectin-1 and galectin-3 to accumulate at
specific spots underneath the plasma membrane, and what
actually causes the formation of exosomes into which these
proteins appear to be packaged in an active fashion.
Other secretory proteins exported
by nonconventional means

HIV-Tat, Herpes simplex VP22 and foamy virus Bet
Besides the classical examples of ER/Golgi-independent
protein secretion described above, a whole variety of
proteins has been reported to be secreted by nonconven-
tional means. Among them are many factors whose
localization-dependent functions, akin to those noted
above, are of tremendous biomedical importance. Such
proteins include virus-encoded factors that are critical for
the viral replication cycle. The most prominent example is
HIV-Tat, one of the auxiliary proteins required by HIV in
addition to structural and enzymatic proteins to replicate its
genome [92]. HIV-Tat has been shown to be released from
both HIV-infected and HIV-Tat-transfected cells in the
absence of appreciable amounts of cell death [93,94].
Intriguingly, HIV-Tat contains a region in its primary
structure termed the basic transduction domain that
appears to enable the protein to traverse membranes
[95,96]. The molecular mechanism of this translocation
process does not seem to involve a proteinaceous machinery
as another HIV-Tat-like protein transduction domain, the
antennapedia third helix domain [96], has been shown to
cross artificial protein-free membranes [97]. Another
unusual feature of protein transduction domains is their
apparent ability to translocate across membranes even at
4 °C [96,98], an observation consistent with a membrane
translocation mechanism independent of proteinaceous
machinery. In all cases, however, protein transduction
domains appear to function in unconventional modes of
protein uptake by mammalian cells. Specifically, in the cases
of HIV-Tat and Herpes simplex tegument protein VP22, it

is rather unlikely that their ER/Golgi-independent export
mechanisms are based on their protein transduction
domains. For example, HIV-Tat secretion from cultured
cells is a temperature-dependent process [94]. Thus, protein
transduction-dependent uptake and ER/Golgi-independent
export of protein transduction domain-containing factors
might be mechanistically distinct processes. Similar to
herpes simplex VP22, a secreted auxiliary protein termed
Bet encoded by foamy viruses [99] has been shown to spread
between cultured cells [100]. Interestingly, both VP22 and
Bet are found in the cytoplasm of expressing cells whereas
they are targeted to the nucleus of cells that received the
2112 W. Nickel (Eur. J. Biochem. 270) Ó FEBS 2003
protein by intercellular spreading [98,100]. In both cases,
this process is not affected by brefeldin A, suggesting that
export of VP22 and Bet from expressing cells does not
involve the ER/Golgi system [98,100]. In conclusion, it
appears likely that uptake by mammalian cells of HIV-Tat,
VP22 and possibly Bet is mediated by transduction domains
in a temperature-insensitive manner whereas export is
mediated in a temperature-sensitive manner by proteina-
ceous machineries that are insensitive to brefeldin A.
Leishmania HASPB
Another quite remarkable example of nonclassical protein
export from eukaryotic cells is the mechanism of cell surface
expression of Leishmania HASPB (hydrophilic acylated
surface protein B) which is found associated with the outer
leaflet of the plasma membrane only in the infectious stages
of the parasite lifecycle [101,102]. The protein is synthesized
on free ribosomes in the cytoplasm and becomes both

myristoylated and palmitoylated at its N-terminus, which
is the molecular basis of how HASPB is anchored in the
membrane [103]. Mutational analysis revealed that an
HASPB construct lacking its 18 N-terminal amino acids is
redistributed into the cytoplasm [103]. The same is true for a
mutant that retains the N-terminus but lacks the myristoy-
lation site [103]. Interestingly, a mutant that lacks the
palmitoylation site but continues to be myristoylated has
been found associated with the cytoplasmic surface of the
Golgi apparatus [103]. Based on these observations, a model
has been proposed in which HASPB is transferred from the
cytoplasm to the outer leaflet of the Golgi membrane, from
where it is transported to the plasma membrane via
conventional vesicular transport. This process would insert
HASPB into the inner leaflet of the plasma membrane. At
present it is completely unclear how HASPB is then
translocated across the membrane, resulting in the insertion
of the two acyl chains in the outer leaflet of the plasma
membrane. Intriguingly, heterologous expression of various
HASPB fusion proteins in mammalian cells revealed the
existence of a machinery that is capable of translocating the
protein across the plasma membrane [103], demonstrating a
conserved pathway among lower and higher eukaryotes. No
endogenous mammalian cargo proteins that make use of
this type of export system have been identified.
Homeodomain-containing transcription factors
and HMG (high mobility group) chromatin-binding
proteins
As another example of nonclassical protein export, two
classes of proteins involved in the overall process of

regulated gene transcription have been proposed to operate
as extracellular factors even though they are normally
localized to the nucleus of mammalian cells [104–106]. For
the transcription factor Engrailed homeoprotein isoform 2
(En2), a potential paracrine signalling activity was postula-
ted as a subpopulation of En2 has been localized to the cell
periphery in caveolae-like structures [105]. In addition, a
small but significant portion of total cellular En2 was found
to reside in membrane-bound vesicles as judged by protease
protection experiments [105]. Therefore, it was reasoned
that En2, despite lacking a conventional ER signal peptide,
might be secreted at a certain rate. This hypothesis was
tested experimentally by coculturing COS cells expressing
the chicken orthologue of En2 (cEn2) with rat primary
neurons demonstrating intercellular transfer of cEn2 [106].
Interestingly, not only export from COS cells but also
import by cocultured primary neurons appears to rely on
nonclassical mechanisms, as cellular uptake of cEn2 was
shown to depend on an unusual WF motif in position 48–49
[106]. This sequence is also required for the uptake of other
homeodomain-containing proteins [107–109]. The internali-
zation of homeodomain-containing proteins apparently
differs from classical endocytosis, as it seems to occur by
direct translocation across the plasma membrane [106]. This
process might be similar to the uptake mechanism of some
viral proteins such as HIV-Tat and Herpes simplex VP22 as
discussed above [108].
About 5% of total cellular En2 becomes externalized by
COS cells which is about the portion that is also found to be
protected against protease treatment. An 11-amino acid

sequence within the homeodomain of En2 has been identified
that, when removed, causes a block in export of the
corresponding mutant protein [106]. This phenotype corre-
lates with the disappearance of the mutant protein from the
protease-protecting organelle, which probably represents a
kind of a secretory compartment [106]. The homeodomain-
derived peptide was later shown to be part of a nuclear export
signal and therefore promotes retrotranslocation of En2
from the nucleus into the cytoplasm [110]. These results have
been taken to mean that retrotranslocation of En2 from the
nucleus to the cytoplasm is a prerequisite for nonclassical
export of En2 [110]. While the homeodomain-derived
peptide was originally thought to represent a signal for
nonclassical export, this view has to be re-evaluated as it
might only trigger cytoplasmic localization of En2 and may
not be required afterwards for externalization of En2.
HMG proteins are intranuclear factors that mediate the
assembly of site-specific DNA-binding proteins within
chromatin [111]. As a surprising finding, but similar to the
homeodomain-containing transcription factors described
above, HMGB1 is secreted during certain physiological
processes such as inflammation. Specifically, monocytes have
been shown to export HMGB1 upon stimulation with
bacterial lipopolysaccharides [112]. Because antibodies
against HMGB1 suppress LPS-induced endotoxemia, and
injection of HMGB1 protein into mice causes toxic shock,
HMGB1 apparently acts as a mediator of endotoxin lethality
in mice [112]. Interestingly, HMGB1 export competence
appears to be a special property of a limited number of cell
types (such as monocytes and macrophages) as many cell

types including lymphocytes are not capable of secreting
HMGB1 [112]. Again, similar to homeodomain-containing
transcription factors, extracellular HMGB1 has also been
shown to act as both an autocrine and paracrine signalling
molecule promoting differentiation processes of the
HMGB1-secreting cell [113,114] or other cells nearby [115].
As with all the examples of unconventional protein
secretion discussed in this review, HMGB1 does not contain
a signal peptide for translocation into the ER [104]. Similar
to IL-1b, FGF-2 and galectin-3 [9], a rise in intracellular
Ca
2+
triggers HMGB1 export [114,115]. Akin to the
mobilization of homeodomain-containing transcription
factors, HMGB1 has been observed to redistribute from
Ó FEBS 2003 Nonclassical protein secretion (Eur. J. Biochem. 270) 2113
the nucleus to the cytoplasm upon activation of monocytes
[116]. A detailed ultrastructural analysis revealed that
redistributed HMGB1 localizes to an endolysosomal
compartment from which secretion can be triggered by
stimuli known to promote lysosomal exocytosis [116]. These
characteristics are strikingly similar to the process of IL-1b
secretion [25]. However, IL-1b secretion from monocytes
can be triggered by adding exogenous ATP, whereas
HMGB1 release is induced by lysophosphatidylcholine.
Moreover, the kinetics of IL-1b and HMGB1 release from
monocytes differ significantly, with IL-1b being secreted
early after monocyte activation and HMGB1 at a later
stage. IL-1b consistently acts at an early phase of inflam-
mation whereas HMGB1 functions as a late mediator of

inflammation (see above). These results have been taken to
indicate that lysosomal exocytosis might involve distinct
populations of endolysosomal vesicles, thereby allowing
different kinetics of cargo release [116].
Direct translocation of proteins from the cytoplasm into
the lumen of lysosomes has been reported [117] but this
pathway appears to function primarily for enhanced
degradation of these proteins [118]. The corresponding
targeting motif KFERQ [118] is not found in the primary
structure of IL-1b, En2 or HMGB1. It therefore appears
more likely that these factors are translocated by a different
mechanism. A potential candidate is ABC1, an ABC
transporter that has been implicated to play a role in the
overall process of IL-1b secretion [27,28].
Cytoplasmic clearance of unfolded proteins
by nonclassical secretion
The mitochondrial matrix protein rhodanese, a monomeric
sulfotransferase, that, following synthesis on free ribosomes
in the cytoplasm, is normally imported into mitochondria,
represents another unusual example of nonclassical protein
export from mammalian cells. When overexpressed in
HEK-293 cells from a strong viral promotor, about 40% of
total rhodanese was found to be secreted into the culture
medium [119]. Export was shown to occur in the absence of
appreciable amounts of cell death and to depend on neither
the mitochondrial targeting sequence of rhodanese nor a
functional ER/Golgi system [119]. Based on the observation
that rhodanese acquires its enzymatic activity only after
import into the mitochondrial matrix (and that the signal
peptide cannot be an inhibitor of enzymatic activity as it is

not cleaved off in the matrix), it was concluded that the
population present in the cytoplasm remains unfolded
before import into mitochondria. Therefore, it has been
postulated that the export pathway detected for rhodanese
represents a mechanism for clearing the cytoplasm of
unfolded proteins that apparently accumulate upon over-
expression [119]. More recently, a potentially similar
example of cytoplasmic clearance of an unfolded protein
population possibly generated by overexpression has been
observed [120]. In this case, an unfolded subpopulation of
transiently overexpressed GFP was found to be secreted in a
brefeldin A-insensitive manner. This effect has not been
observed in stable cell lines that express moderate levels of
GFP in a doxicycline-dependent manner [50]. However,
these different observations are not necessarily inconsistent
as in the latter case an unfolded population of GFP is
unlikely to exist. Interestingly, methylamine and other drugs
known to inhibit nonclassical export of substrates such as
IL-1b, FGF-2, thioredoxin, and the galectins ([9]; see above)
do not block externalization of rhodanese or unfolded GFP
[119,120], again suggesting the existence of distinct mole-
cular mechanisms of unconventional protein secretion.
Targeting motifs and regulation
of nonclassical protein export
In many cases of intracellular protein sorting, short, linear
amino acid sequences have been identified that serve as
sorting motifs, including N-terminal signal peptides for ER
translocation and the N-terminal targeting signals of
mitochondrial proteins [118,121]. Currently, very limited
information is available about motifs directing proteins to

the various pathways of unconventional protein secretion
described above. The most defined one is that of Leishmania
HASPB which consists of a linear sequence of 18 amino
acids at the extreme N-terminus referred to as HASPB-N18
[103]. This sequence is both necessary and sufficient to direct
a corresponding fusion protein to the HASPB export
pathway in both parasites and mammalian cells. HASPB-
N18 is myristoylated at a glycine residue in position 2 and
palmitoylated at a cysteine residue in position 5. HASPB
externalization requires that both residues are acylated.
However, a construct termed HASPB-N10, which contains
both acylation sites but lacks the amino acids 11–18, fails to
translocate across the plasma membrane [103]. These results
suggest that acylation might only be required to initially
insert the protein into the membrane, and the translocation
that follows requires an interaction of the proteinaceous
part of HASPB-N18 with the putative export machinery.
Based on these characteristics, the HASPB export pathway
appears to be unrelated to other examples of nonclassical
protein export described here. As the pathway is functional
in mammalian cells, endogenous substrates are likely to
exist. However, the 18-amino acid sequence found at the
N-terminus of HASPB is not only absent from other
secretory proteins exported by unconventional means but is
also not found in any mammalian protein.
Akin to Leishmania HASPB, the N-terminus of galectin-3
has been proposed to contain targeting information for
nonclassical export [122,123]. When the first 120 amino
acids of galectin-3 are deleted, the residual portion of the
protein is no longer secreted. Conversely, addition of this

N-terminal segment to a cytosolic protein directs the
corresponding fusion protein to the galectin-3 export
pathway. A short sequence comprising residues 89–96
(based on the hamster amino acid sequence) was identified
that, upon deletion, causes a breakdown of galectin-3
export. However, the addition of this small peptide to a
cytosolic protein is not sufficient to direct the resulting
fusion protein to the galectin-3 export pathway suggesting
that, besides the critical role of this short segment, other
determinants for nonclassical export exist in the N-terminal
part of galectin-3 [123]. When compared to the galectin-1
amino acid sequence, no significant homologies can be
found within the N-terminal 120 amino acids of galectin-3.
In contrast to HASPB and galectin-3, the C-terminal half
of FGF-1 has been implicated in its temperature stress-
induced release [124]. A domain comprising a stretch of
2114 W. Nickel (Eur. J. Biochem. 270) Ó FEBS 2003
amino acids from position 83–154 (based on the human
FGF-1 orthologue) appears to prevent the protein from
entering the nucleus, which has been suggested to be a
prerequisite for unconventional export. When the corres-
ponding domain of FGF-2 was transferred to FGF-1,
secretion of the resulting hybrid protein was no longer
observed. These data have been taken to mean that FGF-1
and FGF-2 are exported by distinct pathways [124], which is
consistent with the observation that only FGF-1 release can
be triggered by temperature stress [46,54]. The actual
targeting motifs for nonclassical export have not been
revealedforeitherFGF-1orFGF-2.
For the homeodomain-containing transcription factor

En2, it has been suggested that an 11-amino acid motif
within the homeodomain may function as a signal for
nonclassical export [106]. As discussed above, this
sequence was later found to be part of a nuclear export
signal suggesting that nuclear export of En2 is a
prerequisite for its unconventional secretion [110]. There-
fore, it is rather unlikely that this signal is required for the
export process of En2. Interestingly, En2 has been shown
to be a substrate for protein kinase CK2 which, upon
phosphorylation of En2 within a serine-rich domain,
causes attenuation of En2 secretion [16]. At this point, it is
not clear whether this segment of En2 (residues 146–169)
is part of a signal sequence for nonclassical export or
whether this domain regulates access of En2 to its export
pathway. In either case, the information for En2 export
must lie within the En2 homeodomain, as this part of the
protein alone is an efficient substrate for intercellular
transfer [16]. Phosphorylation-dependent regulation might
be a general principle for the regulation of intercellular
transfer, at least for a subset of these cargo proteins, as it
has also been suggested to play a role in the intercellular
transfer of VP22 [125,126].
Similar to En2 export, many of the proteins described
here are exported in a regulated fashion. For example, IL-1b
and HMGB1 can be released from monocytes upon
stimulation with reagents that induce an inflammatory
response [6,104]. At the same time, En2, IL-1b and HMGB1
are those factors among unconventionally secreted proteins
that appear to be exported from an endosomal subcom-
partment [25,106,116], which might be interpreted as some

kind of storage mechanism from which regulated secretion
of these factors can be triggered. On the other hand, as
discussed above, nonclassical export of for example FGF-1
and galectin-1 are also regulated inducible processes, yet
there is no evidence that these factors are packaged into
intracellular vesicles prior to secretion. The export process
of galectin-1 has been shown to be regulated based on cell
differentiation. For example, during the differentiation of
muscle cells a massive increase of galectin-1 export has been
observed in correlation with the transition from myoblasts
to myotubes [7]. Similarly, galectin-1 export from the
leukemia cell line K-562 can be stimulated by the addition of
differentiation-inducing agents such as erythropoietin [14].
It appears likely that differentiation-correlated galectin-1
export requires the synthesis of specific proteins, which is in
line with the time delay of induced galectin-1 export.
Similarly, FGF-2 export has recently been shown to be
triggered by the expression of the Epstein–Barr virus protein
LMP-1 [15]. This mechanism requires a functional NF-jB
signalling pathway [15], possibly indicating that LMP-1-
mediated stimulation of FGF-2 export involves the induc-
tion of a protein machinery based on de novo synthesis.
Conclusions
As illustrated in Fig. 1, at least four distinct pathways of
unconventional protein secretion exist in mammalian cells
that are fully functional in the absence of an intact ER/
Golgi system. Various fundamental questions arise from
these observations such as why do mammalian cells actually
need additional secretory mechanisms besides the classical
pathway? As noted previously, for the galectins it is

relatively obvious that the alternative secretory pathway
prevents their premature binding to glycolipids and glyco-
proteins within the lumen of the classical secretory pathway
[9]. However, in other cases it is less clear why these cargo
proteins are exported by unconventional means. Other
fundamental questions are: What are the molecular com-
ponents that drive various mechanisms of nonclassical
export? Why do proteins such as HMGB1 with completely
unrelated functions also serve as paracrine signalling
molecules upon unconventional release into the extracellular
space? The answers to these questions are of exceptional
interest as the cargo proteins secreted by unconventional
means are factors whose biological functions are of
tremendous importance to biomedical research. For exam-
ple, FGF-2 has been identified as a major target protein for
the development of antiangiogenic drugs, as it has been
shown that inhibitors of ternary complex formation
between FGF-2 and its high and low affinity receptors
[127] on the surface of target cells display antiangiogenic
activity in vivo [128]. Similarly, unconventional cell-surface
expression of HASPB by Leishmania parasites appears to be
tightly correlated with host cell infection [101,102] and
therefore the HASPB export pathway might be an excellent
target for the development of drugs against tropical and
subtropical diseases termed the leishmanias. These path-
ways are in general attractive targets, because it may be
possible to identify inhibitors that do not interfere with the
essential function of the classical secretory pathway. There-
fore, elucidation of the molecular machineries controlling
the various kinds of nonclassical export might provide a

whole variety of novel target proteins suitable for drug
design.
So far, a biochemical analysis of the molecular machi-
neries of nonclassical protein export has proven difficult
because in many cases the export process is relatively
inefficient. Also, it is not yet clear whether unfolding of the
various cargo proteins is required for unconventional
secretion. If so, classical methods employing dihydrofolate
reductase [129] domains to trap cargo proteins at the site of
translocation could be used in order to allow a biochemical
analysis of the export apparatus. It is also a major problem
that, in most cases, only very limited information is
available about the motifs that target cargo proteins to
the nonclassical export routes. Recently, novel assays have
been developed which deploy fluorescence activated cell
sorting to reconstitute unconventional protein export path-
ways on a quantitative basis [50,79]. This new approach
might facilitate genetic screens in mammalian cells and
is compatible with systematic high throughput screening
Ó FEBS 2003 Nonclassical protein secretion (Eur. J. Biochem. 270) 2115
technologies for the identification of low molecular mass
inhibitors of the processes described here. Thus, the
elucidation of the molecular mechanisms of nonclassical
protein export from eukaryotic cells will not only solve a
fundamental problem in current cell biology but will also
lead to the identification of novel target proteins, with great
value for biomedical research.
Acknowledgements
I would like to thank Britta Bru
¨

gger (Biochemie-Zentrum Heidelberg),
Tracy LaGrassa (Biochemie-Zentrum Heidelberg), Blanche Schwap-
pach (Zentrum fu
¨
r Molekulare Biologie Heidelberg), Ju
¨
rgen Bernhagen
(University Hospital RWTH Aachen) and Markus Ku
¨
nzler (ETH
Zu
¨
rich) for critical comments on the manuscript, as well as all members
of my laboratory for helpful discussions. Work in the laboratory of the
author is supported by grants from the German Research Foundation
(DFG) and the Ministry of Science, Research and the Arts of the State
of Baden-Wu
¨
rttemberg.
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