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

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:

[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

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

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+

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

[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].

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

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

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

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

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

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

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

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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