TALKING POINT
secreted by cells and plays at least four
Moonlighting proteins
Constance J. Jeffery
The idea of one gene one protein one function has become too simple
because increasing numbers of proteins are found to have two or more dif-
ferent functions. The multiple functions of such moonlighting proteins add
another dimension to cellular complexity and benefit cells in several ways.
However, cells have had to develop sophisticated mechanisms for switch-
ing between the distinct functions of these proteins.
AS THE SEQUENCES of complete gen-
omes rapidly become available, the next
step is to determine the functions of the
encoded proteins and how all these pro-
teins come together to make a living cell.
Many protein functions can be inferred
from the known functions of homologous
proteins, but the existence of multifunc-
tional proteins complicates such interpre-
tation, as well as providing a fascinating
window into the complex interactions
among the various components that make
up a modern cell. As in the case of phos-
phoglucose isomerase (described below),
a protein can be a key enzyme in gly-
colysis and then be found moonlighting
outside the cell as a nerve growth factor.
Here, I discuss examples of moon-
lighting proteins that demonstrate the
variety of proteins that have multiple
roles (see Table 1), the different mecha-
nisms for switching between functions,
and ways in which moonlighting can
benefit the cell. Even though I am limit-
ing the definition of moonlighting pro-
teins and excluding proteins that are
the result of gene fusions, homologous
but non-identical proteins, splice vari-
ants, proteins whose post-translational
modifications can vary and proteins
that have a single function but can oper-
ate in different locations or utilize differ-
ent substrates the examples given
below are by no means a complete list.
Increasingly, we are finding that gene
products of higher organisms moonlight.
Mechanisms
The function of a moonlighting protein
can vary as a consequence of changes in
cellular localization, cell type, oligomeric
state, or the cellular concentration of a
ligand, substrate, cofactor or product
C. J. Jeffery is at the Rosenstiel Center,
MS029, Brandeis University, Waltham,
MA 02454-9110, USA.
Email: cjeffery@alum.mit.edu
8 09680004/99/$ See front matter 1999, Elsevier Science. All rights reserved.
TIBS 24 JANUARY 1999
additional roles. It is the same protein as
neuroleukin3,4, which is both a cytokine
that causes B cells to mature into
antibody-secreting cells5, and a nerve
growth factor that promotes the sur-
vival of some embryonic spinal neurons
and some sensory nerves6. In addition,
phosphoglucose isomerase/neuroleukin
is the same protein as autocrine motility
factor (AMF), which is a cytokine that
stimulates cell migration7. Finally, it is also
a differentiation and maturation mediator
(DMM) that can cause differentiation of
human myeloid leukemia cells8.
Thymidine phosphorylase also has dif-
(Fig. 1). These different mechanisms are ferent functions inside and outside the
not mutually exclusive and, in many cell9. In the cell cytoplasm, it catalyzes
cases, a protein uses a combination of the dephosphorylation of thymidine,
methods to switch between functions. deoxyuridine and their analogs to their
Differential localization. The same protein bases and 2-deoxyribose 1-phosphate.
can perform two different functions in In the extracellular fluid, it is platelet-
two different locations within the cell. derived endothelial cell growth factor,
The Escherichia coli PutA protein has which stimulates endothelial cell growth
proline dehydrogenase and pyrroline-5- and chemotaxis.
carboxylate dehydrogenase activity Differential expression. The same protein
when it is associated with the plasma can also perform different functions
membrane, but lacks enzymatic activity when it is expressed in different cell types.
and binds DNA as a transcriptional re- Neuropilin is a cell surface receptor on
pressor when it is in the cytoplasm1,2. endothelial cells that detects vascular
One function within the cell and another outside. endothelial growth factor and indicates
Other proteins can have one function when new blood cells are needed10. In
within the cell and another outside it. nerve axons, it is again a cell surface re-
Phosphoglucose isomerase is a ubiqui- ceptor but detects a different ligand,
tous cytosolic enzyme: it catalyzes the semaphorin III, and helps steer axons to
second step in glycolysis, the intercon- their proper destinations.
version of glucose 6-phosphate and Oligomerization. Some proteins have
fructose 6-phosphate. However, it is also one enzymatic activity as a monomer
Table 1. Examples of moonlighting enzymes
One function Additional functions Refs
PutA proline dehydrogenase Transcriptional repressor 1, 2
Phosphoglucose isomerase Neuroleukin, autocrine motility factor, differentiation 3, 8
and maturation mediator
Thymidine phosphorylase Platelet-derived endothelial cell growth factor 9
Neuropilin (VEGF receptor) Receptor for semaphorin III (nerve axons) 10
Uracil-DNA glycosylase Glyceraldehyde-3-phosphate dehydrogenase 11
Aconitase Iron-responsive-element binding protein (IRE-BP) 12, 15
Carbinolamine dehydratase Dimerization cofactor (DCoH) 16
Escherichia coli thioredoxin Subunit of T7 DNA polymerase 17
E. coli aspartate receptor Maltose-binding-protein receptor 13, 14
PMS2 mismatch-repair enzyme Hypermutation of antibody variable chains 24
Ribosomal proteins DNA repair, translational regulators, development 25
regulators, etc.
Lens crystallins Heat-shock proteins, lactate dehydrogenase, 26
argininosuccinate, retinaldehyde dehydrogenase,
lyase, enolase, quinone oxidoreductase,
glyceraldehyde-3-phosphate dehydrogenase, etc.
CFTR chloride channel Regulator of other epithelial anion channels 18
P-glycoprotein (transporter) Regulator of cell-swelling ion channel 19, 20
Thrombin protease Ligand for cell surface receptors 21
Thymidylate synthase Translation inhibitor 22
E. coli birA biotin synthetase Bio operon repressor 23
Mitochondrial LON protease Chaperone 27
Bacterial FtsH chaperone Metalloprotease 27
Band-3 anion exchanger Regulator of glycolysis 28
PII: S0968-0004(98)01335-8
TIBS 24 JANUARY 1999 TALKING POINT
and another as a multimer.
The 37-kDa subunit of human (a) (b)
glyceraldehyde-3-phosphate de-
hydrogenase, for example, is a
glycolytic enzyme that, as a
tetramer, converts glyceralde- 1
2
hyde 3-phosphate to 1,3-diphos- 1 2
phoglycerate. As a monomer, it
is a nuclear uracil-DNA glyco-
sylase11. This activity is impor-
tant for removing uracil that is
Different locations within the cell Inside and outside the cell
present in DNA because of ac-
cidental use of dUTP during
DNA synthesis or deamination (c)
of cytosine residues.
Ligand/substrate concentration.
The function of an enzyme can
be switched by the amount of
2
substrate, ligand or cofactor 1
available. The PutA protein
mentioned above binds to the
membrane when substrate (pro-
line) concentrations are high but Expression by different cell types
binds to DNA when substrate
concentrations decrease1. The
oligomeric state of glyceralde- (d) (e)
hyde-3-phosphate dehydrogen-
ase (nuclear uracil-DNA gly-
cosylase) is influenced by 1 2 1 2
cellular concentrations of ATP,
Binding of a cofactor Oligomerization
NAD1 and protein11. Aconitase
is an iron-dependent enzyme
that has catalytic activity only
when cellular iron concen- (f) (g)
trations are high. When the
iron concentration decreases,
the aconitase protein loses its
4Fe4S FeS cluster and its en- 1 2 1 2
zymatic activity, and takes on Complex formation Multiple binding sites
a new role as the iron respon-
sive element (IRE)-binding
protein (IRE-BP)12. By binding Figure 1
to a stem-loop, the IRE, in the Examples of mechanisms for switching between functions (1 and 2). (a) A protein can have different
functions in different locations within a cell (e.g. when bound to the cell membrane as opposed to
59 untranslated region of fer- DNA). (b) Proteins can have enzymatic activity in the cell cytoplasm but serve as growth factors when
ritin mRNA, the IRE-BP pro- they are secreted. (c) Proteins can have different functions when they are expressed by different cell
motes the synthesis of ferritin. types (e.g. an endothelial cell as opposed to a neuron). (d) Binding of substrate, product or a cofactor
In addition, it binds to several can cause a switch in activity. (e) A multimer can have an activity that differs from that of the monomer.
IREs in the 39 untranslated re- (f) Interaction with different polypeptides to form different multisubunit complexes can result in a
gion of the transferrin receptor switch in function. (g) Some proteins can have different binding sites for different substrates.
mRNA and controls the degra-
dation of transferrin receptor mRNA. motility factor response, the enzyme to the quinonoid dihydrobiopterin. This
The aconitase/IRE-BP protein is either might use its active site for binding to its protein is identical to the dimerization
an enzyme or an RNA-binding protein, receptor when functioning as a cyto- cofactor DCoH, which regulates the
not both simultaneously. kine7. Some evidence also indicates that DNA-binding activity of a homeodomain
Binding sites. Moonlighting enzymes the aconitase active site is the surface transcription factor, hepatic nuclear fac-
can perform their multiple functions by used for RNA binding when the protein tor 1a (HNF-1a), by affecting its dimeriz-
using distinct binding sites. The E. coli functions as the IRE-BP (Ref. 15). ation16. Another interesting example is
aspartate receptor, which functions in Complex formation. Some polypeptide E. coli thioredoxin, which is important
bacterial chemotaxis, is also a maltose- chains form parts of different multi- in deoxyribonucleotide synthesis: it helps
binding protein (MBP) receptor. It has subunit protein complexes that have dif- reduce ribonucleoside diphosphates to
different but overlapping binding sites ferent roles. One example is carbinol- deoxyribonucleoside diphosphates. This
for aspartate and MBP13,14. Given that in- amine dehydratase, an important enzyme protein has been recruited by T7 phage,
hibitors of phosphoglucose isomerase in liver phenylalanine metabolism, which in which it functions as a subunit of a
catalytic activity block the autocrine converts 4a-hydroxytetrahydrobiopterin heterodimeric DNA polymerase17.
9
TALKING POINT
Regulation of proteins that have similar func-
tions or of multiple parts of a pathway. Some
proteins have one activity and, in ad-
dition, serve as a regulator of other pro-
teins that perform similar functions.
The cystic fibrosis transmembrane-
conductance regulator (CFTR) is both a
cAMP-dependent chloride channel and
a regulator of an epithelial sodium chan-
nel (ENaC)18. Similarly, the multidrug-
resistance P-glycoprotein transmembrane
transporter regulates ion channels that
respond to cell swelling19,20. Some pro-
teins sense a change in the organism
and then interact with different macro-
molecules to trigger multiple pathways as
part of an overall response. Thrombin is
a protease that cleaves fibrinogen to
produce fibrin in the blood-clotting cas-
cade. It is also a ligand for the G-protein-
coupled receptor PAR-1 (Ref. 21). Acti-
vation of this receptor causes platelets
to stick together.
Self-regulation of transcription or translation.
Many biosynthetic or catabolic enzymes
regulate their own transcription or trans-
lation on the basis of the availability of
enzyme, substrate or product. Thymidyl-
ate synthase, for example, binds to a
stem-loop structure in the 59 untrans-
lated region of its own mRNA and in-
hibits translation22. When substrate con-
centration is limiting, the PutA protein
binds to DNA and prevents transcrip-
tion of the putA gene. The product of the
E. coli birA gene is biotin-holoenzyme
10
synthetase, which synthesizes biotinyl
59-adenylate. When there is sufficient
biotinyl 59-adenylate in the cytoplasm,
the protein represses the Bio operon23.
Evolution of moonlighting proteins
The more extreme examples of moon-
lighting raise two questions. (1) How did
moonlighting arise during evolution? (2)
How does it benefit the cell? The sim-
plest answer is that organisms evolve by
making use of whatever is available. Sev-
eral multifunctional proteins are ubiqui-
tous enzymes for example, the two
glycolytic enzymes mentioned above.
These glycolytic enzymes are present in
eukaryotes, eubacteria and archea, and
have probably been around for over a
billion years ample time for additional
uses to be made of them. The mouse
PMS2 gene is a ubiquitous mismatch-
repair enzyme that appears to have been
recruited by some blood cell types for
use in somatic hypermutation of anti-
body variable chains24. Many lens crys-
tallins and some ribosomal proteins are
identical to cytoplasmic proteins and
were apparently recruited during the
evolution of the eye and the ribosome,
respectively25,26.
In terms of protein structure, a new
function can be a result of new uses for
an existing binding site or modifications
of unused regions of the protein struc-
ture. Many enzymes appear to be much
larger than is necessary for performing
TIBS 24 JANUARY 1999
a single function that requires a single
binding site. They often have large areas
of apparently unused solvent-exposed
surface and many pockets on the pro-
tein surface that could be modified to
make additional binding sites. As long as
these additional functions do not inter-
fere with the original function of the
protein, they might benefit the cell.
For a protein that has multiple related
functions, the additional roles might be
due to low specificity of an active site. In
some cases, they could have arisen by a
broadening of the specificity of an ances-
tral active site so that the protein can
now catalyze reactions that involve mul-
tiple, apparently unrelated substrates or
bind to multiple binding partners. (Of
course, examples of less extreme cases
where a protein interacts with two or
more similar ligands or substrates are
quite common.) Such proteins might
have evolved through mutations in the
active site or use of regions surrounding
the active site. The opposite might also
be true for other enzymes: an ancestral
enzyme might have been able to bind
many ligands, and its specificity might
have failed to increase during evolution.
There are several ways in which multi-
functional proteins can benefit an organ-
ism and result in a competitive advantage
during evolution. The simplest reason
at least in prokaryotes is that, by
having multifunctional proteins, an or-
ganism has fewer proteins to synthesize
 TIBS 24 JANUARY 1999
and, consequently, less DNA to replicate.
This would save a great deal of energy in
growth and reproduction.
Moonlighting proteins can also provide
a means of coordinating cellular activ-
ities. The regulation of EnaC channel ac-
tivity by the CFTR channel (see above)
is probably important for the mainte-
nance of epithelial ion homeostasis.
Similarly, enzymes that affect the activ-
ity of other enzymes that catalyze simi-
lar reactions provide a means of joint
regulation. Biosynthetic or catabolic en-
zymes that regulate their own transcrip-
tion or translation on the basis of the
availability of enzyme, substrate or prod-
uct provide a sensitive and direct feed-
back mechanism that ensures that cells
make only as much enzyme as they need.
In addition, proteins that trigger multiple
related pathways, such as thrombin, allow
a more effective overall response than
would the individual functions alone.
In the cases of other proteins, the
different functions are complementary
and serve as switches between path-
ways. For example, several proteases
also have chaperone activity27. Eukary-
otic (rat liver and yeast) mitochondrial
LON is an ATP-dependent protease that
degrades some mitochondrial proteins,
and is also a mitochondrial chaperone.
Bacterial FtsH protein assists in the
transport of proteins into and across
membranes, and is an ATP-dependent
metalloprotease. The yeast mitochon-
drial homologs of FtsH (Afg3p and
Rca1p) facilitate subunit assembly of
F1F0-ATPase and also have protease ac-
tivity. These two functions might be
linked: Afg3p/Rca1p could either help
to fold or degrade the bound polypep-
tide chain depending on factors such
as whether or not the chain is par-
tially folded.
In the above examples, there appears
to be a relationship between the multiple
functions of each protein; however, for
some proteins, there is currently no clear
connection between their functions. The
band 3 protein of the red blood cell
plasma membrane is best known as an
anion exchanger but also regulates the
rate of glycolysis28. Its cytoplasmic N-
terminal domain binds to aldolase, glycer-
aldehyde-3-phosphate dehydrogenase
and phosphofructokinase, and causes a
decrease in their activities and an accu-
mulation of glycolytic intermediates.
Conclusions
The increasing number of proteins
that are being found to moonlight adds
another level to our understanding of
cellular complexity. There is a great deal
of interaction between macromolecules,
and the modern cell is a sophisticated
and highly organized network. Moon-
lighting proteins provide one way of
coordinating cellular activities.
The number of moonlighting proteins
also has implications for prediction of
the functions and interactions of the
many proteins whose sequences are be-
coming available (through the recent
successes of the genome projects): it
adds to the difficulty of interpreting gen-
ome sequences. In addition, because
many proteins can have multiple inter-
actions, it could be difficult to ascertain
who interacts with whom in the cell.
Acknowledgement
I thank Greg Petsko for critical
reading of the manuscript and helpful
suggestions.
TALKING POINT
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