Báo cáo khoa học: A Caenorhabditis elegans model of orotic aciduria reveals enlarged lysosome-related organelles in embryos lacking umps-1 function potx

A Caenorhabditis elegans model of orotic aciduria reveals
enlarged lysosome-related organelles in embryos lacking
umps-1 function
Steven Levitte
1
, Rebecca Salesky
1
, Brian King
2
, Sage Coe Smith
2
, Micah Depper
1
, Madeline Cole
2
and Greg J. Hermann
1,2
1 Department of Biology, Lewis & Clark College, Portland, OR, USA
2 Program in Biochemistry and Molecular Biology, Lewis & Clark College, Portland, OR, USA
Introduction
Lysosome-related organelles (LROs) represent a
diverse collection of specialized compartments that
share features in common with conventional lysosomes
[1–3]. LROs perform a variety of important physiologi-
cal functions. In mammals, for example, lamellar
bodies function in the storage and release of lung
Keywords
gut granule; lysosome-related organelle;
orotic aciduria; UMPS
Correspondence
G. Hermann, Department of Biology, Lewis

& Clark College, 0615 S.W. Palatine Hill Rd,
Portland, OR 97219, USA
Fax: +1 503 768 7658
Tel: +1 503 768 7568
E-mail:
(Received 16 October 2009, revised 26
December 2009, accepted 5 January
2010)
doi:10.1111/j.1742-4658.2010.07573.x
Gut granules are cell type-specific lysosome-related organelles found within
the intestinal cells of Caenorhabditis elegans. To investigate the regulation
of lysosome-related organelle size, we screened for C. elegans mutants with
substantially enlarged gut granules, identifying alleles of the vacuolar-type
H
+
-ATPase and uridine-5¢-monophosphate synthase (UMPS)-1. UMPS-1
catalyzes the conversion of orotic acid to UMP; this comprises the two ter-
minal steps in de novo pyrimidine biosynthesis. Mutations in the ortholo-
gous human gene UMPS result in the rare genetic disease orotic aciduria.
The umps-1()) mutation promoted the enlargement of gut granules to 250
times their normal size, whereas other endolysosomal organelles were not
similarly affected. UMPS-1::green fluorescent protein was expressed in
embryonic and adult intestinal cells, where it was cytoplasmically localized
and not obviously associated with gut granules. Whereas the umps-1())
mutant is viable, combination of umps-1()) with mutations disrupting gut
granule biogenesis resulted in synthetic lethality. The effects of mutations
in pyr-1, which encodes the enzyme catalyzing the first three steps of de
novo pyrimidine biosynthesis, did not phenotypically resemble those of
umps-1()); instead, the synthetic lethality and enlargement of gut granules
exhibited by the umps-1()) mutant was suppressed by pyr-1()).Ina

search for factors that mediate the enlargement of gut granules in the
umps-1()) mutant, we identified WHT-2, an ABCG transporter previously
implicated in gut granule function. Our data suggest that umps-1()) leads
to enlargement of gut granules through a build-up of orotic acid. WHT-2
possibly facilitates the increase in gut granule size of the umps-1()) mutant
by transporting orotic acid into the gut granule and promoting osmotically
induced swelling of the compartment.
Abbreviations
DAPI, 4¢,6-diamidino-2-phenylindole; DIC, differential interference contrast; GFP, green fluorescent protein; HPS, Hermansky–Pudlak
syndrome; LRO, lysosome-related organelle; ODC, orotidine-5¢-monophosphate decarboxylase; OMP, orotidine 5¢-monophosphate; OPRT,
orotate phosphoribosyltransferase; RNAi, RNA interference; UMPS, uridine-5¢-monophosphate synthase; V-ATPase, vacuolar-type
H
+
-ATPase.
1420 FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS
surfactant, and melanosomes act to synthesize and
store body pigments [2,4]. Investigation of the genetic
basis of Hermansky–Pudlak syndrome (HPS), which is
characterized by defects in the formation and function
of LROs, has led to the identification of 15 genes act-
ing in the trafficking pathways to LROs [5].
Although mutations in HPS genes typically result in
a reduced number of LROs, there is a subset of HPS
mutations that additionally promote the formation of
dramatically enlarged LROs, including melanosomes
[6,7] and lamellar bodies [8,9]. Lamellar bodies are
similarly enlarged in Tangier disease, which results
from defects in the function of the ABC transporter
ABCA1 [10]. Most dramatically, nearly every class of
LRO is enlarged in patients with Chediak–Higashi syn-

drome [11]. In none of these diseases do we clearly
understand the mechanistic basis for LRO enlarge-
ment, reflecting our lack of insight into the processes
that control the stereotypic size and morphology of
LROs.
Gut granules are intestinal cell-specific LROs found
in the nematode Caenorhabditis elegans. In addition to
typical lysosomal characteristics, gut granules stain
with Nile Red, a marker for hydrophobic material,
and contain birefringent and autofluorescent materials,
which are uniquely localized to the gut granule
[12–16]. Gut granule formation is initiated during early
embryogenesis, soon after endoderm specification, and
intestinal cells typically contain hundreds of gut
granules [12–14]. Gut granule biogenesis requires the
activity of conserved genes that function generally in
LRO formation, including those encoding the HOPS
complex, the AP-3 complex, the ABC transporter
PGP-2, and the Rab GTPase GLO-1 [13,15].
Here we describe the results of a genetic screen to
identify factors involved in regulating gut granule size,
and present a phenotypic, cellular and molecular
characterization of one of these genes, umps-1.
Results
A screen for mutants with enlarged gut granules
in embryonic intestinal cells
Gut granules are abundant, cell type-specific, LROs
that are present within the intestinal cells of C. elegans
embryos, larvae, and adults [12,13]. The formation of
gut granules is initiated during early embryogenesis,

and is directly controlled by the regulatory program
governing intestinal cell fate and differentiation in the
early C. elegans embryo [14,17,18]. We have been
investigating the mechanisms controlling the assembly
and morphology of gut granules during embryogenesis
in order to identify the primary regulators of these
processes.
In adult C. elegans intestinal cells, the enlargement
of endolysosomal organelles typically results in a Vac
(vacuolated appearance) phenotype, characterized by
the presence of cytoplasmic vacuoles when visualized
with differential interference contrast (DIC) micros-
copy. The vacuolization of the adult intestine is associ-
ated with enlargement of early endosomes [19],
recycling endosomes [20], and late endosomes ⁄ lyso-
somes [21,22]. We reasoned that enlargement of gut
granules would similarly result in a Vac phenotype.
We first analyzed strains known to exhibit enlarged
endolysosomal compartments in adult or embryonic
intestinal cells for vacuolization of the embryonic
intestine. Only one of the mutants, ppk-3()), displayed
an embryonic Vac phenotype (Fig. 1D,E; Table 1). We
therefore performed a screen for additional mutants
that contained vacuoles within embryonic intestinal
cells.
We identified seven mutants exhibiting a Vac pheno-
type. Complementation tests and molecular cloning
showed that these mutants were defective in three
genes: ppk-3 (one allele), unc-32 (five alleles), and
umps-1 (one allele). The ppk-3()) mutant displayed

prominent vacuoles in the intestine (Fig. 1D,E;
Table 1), as well as in other embryonic cells, as has
been reported previously [21]. The ppk-3 gene encodes
a phosphatidylinositol-3-kinase that catalyzes the for-
mation of phosphatidylinositol 3,5-bisphosphate and is
orthologous to PIKfyve in mammals and Fab1p in
yeast [21]. Cells lacking the function of these kinases
display dramatically enlarged late endolysosomal com-
partments [23]. The unc-32()) and umps-1()) mutants
contained vacuoles exclusively within embryonic intes-
tinal cells (Fig. 1G,H,J,K; Table 1). The unc-32 gene
encodes an intestinally expressed V
0
subunit of the
vacuolar-type H
+
-ATPase (V-ATPase) [24,25]. The
V-ATPase associates with embryonic gut granules [13],
where it functions in acidification [26]. The umps-1
gene encodes UMP synthase (UMPS), which is pre-
dicted to function in de novo pyrimidine biosynthesis
[27].
We analyzed whether gut granules were enlarged in
the vac mutants. Embryonic gut granules contain bire-
fringent material [13,14] and the integral membrane
ABC transporter PGP-2 [15]. The vacuoles within
ppk-3()) intestinal cells did not contain birefringent
material (Fig. 1D,E; Table 1). Although PGP-2-marked
gut granules were slightly enlarged in ppk-3()) embryos
(Fig. 1F; Fig. S1), they did not match the size of vacu-

oles present within ppk-3()) embryos (Fig. 1D,F).
These observations indicate that the vacuoles visible in
S. Levitte et al. UMPS-1 and gut granule size
FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS 1421
ppk-3()) embryos by DIC microscopy are not gut
granules. The ppk-3()) adults displayed a slight
enlargement of autofluorescent gut granules (Fig. S1).
Thus, ppk-3 plays only a minor role in regulating gut
granule size. In contrast, the vacuoles within unc-32())
and umps-1()) embryos contained birefringent material
(Table 1; Fig. 1G,H,J,K), and dramatically enlarged
PGP-2-containing compartments were present in
these mutants (Fig. 1I,L), consistent with gut granule
enlargement. Here, we present our analysis of the role
that UMPS-1 plays in gut granule formation and
morphology; detailed studies of the role that the V-AT-
Pase plays in regulating these processes will be described
elsewhere.
Disrupting the activity of umps-1, a gene that
functions in pyrimidine biosynthesis, leads to a
Vac phenotype
We identified umps-1 as the gene disrupted in the vac
mutant zu456 (see Experimental procedures). Promi-
nent vacuoles were present within the intestinal cells of
umps-1(zu456) embryos from the ‘lima bean’ stage
through to hatching (Fig. 1J; Fig. S2). Vacuoles dimin-
ished in size and number during the L1 stage, and
L2-stage to adult-stage animals exhibited normal intes-
tinal morphology (Fig. S2). umps-1(RNAi) led to an
embryonic Vac phenotype that was indistinguishable

from that caused by umps-1(zu456) (Table 1). Despite
the dramatic vacuolization of the embryonic intestine,
umps-1(zu456) animals can be maintained as a homo-
zygous line.
UMPS-1 is orthologous to mammalian UMPS [27],
a bifunctional enzyme that catalyzes the two terminal
reactions in de novo pyrimidine biosynthesis [28]
(Fig. 2A,C). The orotate phosphoribosyltransferase
(OPRT) activity of UMPS promotes the conversion of
orotic acid to orotidine 5¢-monophosphate (OMP).
The OMP decarboxylase (ODC) activity of UMPS
catalyzes the formation of UMP from OMP. The
C. elegans UMPS-1 protein exhibits both OPRT and
ODC activity in vitro [27]. The sequence of umps-1
from zu456 showed a mutation that destroys the pre-
dicted translation initiation site (Fig. 2B). Use of the
next downstream ATG would result in the formation
of a short, out-of-frame peptide. We therefore con-
clude that zu456 is probably a null allele of umps-1.
The C. elegans gene R12E2.11 codes for a protein
homologous to the OPRT domain of human and
C. elegans UMPS. In vitro, R12E2.11 has OPRT activ-
ity but lacks ODC activity [27], suggesting that it
might functionally overlap with UMPS-1.
R12E2.11(RNAi) did not result in the formation
of embryonic vacuoles (Table 1). In addition,
R12E2.11(RNAi) did not obviously alter the forma-
tion and size of embryonic vacuoles in umps-1(zu456)
A
B

C
D
E
F
G
H
I
JK
L
DIC Polarization PGP-2
Wild typeppk-3 (n2668)unc-32 (f123)umps-1 (zu456)
Fig. 1. Analysis of embryonic vac mutants
for gut granule enlargement. Prominent
vacuoles visible with DIC microscopy that
were lacking in wild type (A) were present
within the intestinal cells of ppk-3()) (D),
unc-32()) (G) and umps-1()) (J) embryos.
The vacuoles (white arrowheads) (D) in
ppk-3()) embryos did not contain birefrin-
gent material (white arrows) (E), as they did
in unc-32()) and umps-1()) embryos (G, H,
J, K). (C, F, I, L) PGP-2 staining (marked by
white arrows) in pretzel-stage embryos.
PGP-2-labeled compartments in ppk-3())
embryos (F) were slightly enlarged in
comparison with the wild type (C). In
contrast, PGP-2-containing compartments
were dramatically enlarged in unc-32()) (I)
and umps-1()) (L) embryos. The intestine is
flanked by black arrowheads. Embryos are

approximately 50 lm in length.
UMPS-1 and gut granule size S. Levitte et al.
1422 FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS
embryos (Table 1) or the persistence of intestinal vacu-
oles in umps-1(zu456) larvae (data not shown), sug-
gesting that R12E2.11 does not play a major role in
regulating the size of intestinal organelles. Moreover,
R12E2.11(RNAi) did not result in phenotypes charac-
teristic of defects in pyrimidine biosynthesis, suggesting
that it is not essential for this process (Table S1 and
data not shown).
The umps-1(zu456) line, while being viable, exhib-
ited partially penetrant embryonic and larval lethality.
Fifty-six per cent of umps-1(zu456) embryos failed to
hatch (Table S2). In addition, 30% of umps-1(zu456)
Table 1. Vacuole formation in embryonic intestinal cells. All strains were grown at 22 °C. Pretzel-stage embryos were scored using DIC
microscopy for the presence of vacuoles in embryonic intestinal cells. Large vacuoles were typically ‡ 1.5 lm, and small vacuoles were
between 0.8 and 1.4 lm in diameter. Polarization microscopy was used to assess the presence of birefringent material within vacuoles.
n, number of embryos scored.
Genotype
Percentage of
embryos with large
vacuoles containing
birefringent material
Percentage of embryos
with large vacuoles
lacking birefringent
material
Percentage of
embryos with small

vacuoles containing
birefringent material n
Wild type 0 0 0 418
Wild type + 5 mgÆmL
)1
uracil 0 0 0 55
Enlarged endolysosomal compartments
alx-1(gk275) 00 0 70
cup-5(zu223)
a
00 0 31
ppk-3(n2668) 0 100 0 59
ppk-3(ok1150)
b
032 0 92
ppk-3(zu443) 0 100 0 90
rab-10(dx2) 00 0 53
rme-1(b1045) 00 0 36
tat-1(kr15) 00 0 54
V-ATPase
unc-32(f121)
c
26 0 0 125
unc-32(f123)
c
28 0 0 68
De novo pyrimidine biosynthesis
umps-1(zu456) 100 0 0 > 2000
umps-1(zu456) +5mgÆmL
)1

uracil 96 0 4 73
umps-1(zu456) ⁄ umps-1(+)
d
00 0 50
umps-1(zu456) · umps-1(+)
e
100 0 0 30
umps-1(RNAi)
f
75 0 16 227
pyr-1(cu8) 00 0 58
pyr-1(RNAi)
f
00 0 32
R12E12.11(RNAi)
f
00 0 52
Transgenic rescue
g
umps-1(zu456)+ WRM0627dD02 53 0 0 43
umps-1(zu456)+ UMPS-1::GFP 0 0 0 31
Double mutants
h
umps-1(zu456); apt-6(ok429)
i
0 0 27 59
umps-1(zu456); glo-1(zu437)
j
00 0 22
umps-1(zu456); mrp-4(ok1095) 57 43 0 54

umps-1(zu456); pgp-2(kx55)
k
81 0 19 90
umps-1(zu456); wht-2(ok2775)
l
63 0 17 104
umps-1(RNAi); wht-2(ok2775)
f
80 8 51
umps-1(zu456); wht-2(RNAi)
f
36 0 0 42
umps-1(zu456); pyr-1(RNAi)
f
10 0 73
umps-1(RNAi); pyr-1(cu8)
f
00 0 31
umps-1(zu456); pyr-1(cu8)
m
00 0 40
umps-1(zu456); R12E2.11(RNAi)
f
100 0 0 31
Mosaic RNAi
rrf-1(pk1471)
n
00 0 30
rrf-1(pk1471); umps-1(RNAi) 69 0 20 55
S. Levitte et al. UMPS-1 and gut granule size

FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS 1423
L1-stage larvae did not reach adulthood (Table S2).
We found that the overall rate of embryogenesis was
delayed in umps-1(zu456) embryos; however, all of the
major tissues appeared to be properly specified and to
differentiate normally, and there were no obvious
developmental defects in umps-1(zu456) embryos prior
to the bean stage (data not shown). To determine
when embryogenesis was affected in umps-1())
embryos, we monitored the development of individual
bean-stage umps-1(zu456) and wild-type embryos.
Thirty-five per cent (n = 49) of umps-1(zu456)
embryos elongated four-fold, whereas 100% of
Table 1. (Continued)
Genotype
Percentage of
embryos with large
vacuoles containing
birefringent material
Percentage of embryos
with large vacuoles
lacking birefringent
material
Percentage of
embryos with small
vacuoles containing
birefringent material n
rde-1(ne219); [elt-2p::rde-1(+)]
n
00 030

rde-1(ne219); [elt-2p::rde-1(+)]; umps-1(RNAi) 00 040
a
Embryos scored were the progeny of cup-5(zu223) unc-36(e251) adults derived from a cup-5(zu223) unc-36(e251) ⁄ qC1 line.
b
Embryos
scored were the progeny of + ⁄ szT1[lon-2(e678)]; ppk-3(ok1150) ⁄ szT1 adults. Twenty-five per cent of the embryos were predicted to be ppk-
3()) ⁄ ppk-3()).
c
The unc-32 alleles analyzed result in zygotic lethality. Therefore, the embryos scored were the progeny of dpy-17(e164) unc-
32()) ncl-1(e1865) ⁄ qC1 dpy-19(e1259) glp-1(q339) adults. Twenty-five per cent of the embryos were predicted to be unc-32()) ⁄ unc-32()).
The linked dpy17(e164) ncl-1(e1865) markers did not result in a vacuole phenotype.
d
The embryos scored were the progeny of umps-
1(+) ⁄ umps-1()) adults. Twenty-five per cent of the embryos were expected to be umps-1()) ⁄ umps-1()).
e
umps-1(+); mIs11[GFP] males
were mated with umps-1(zu456) hermaphrodites, and outcross umps-1()) ⁄ umps-1(+) embryos were recognized by their GFP expression and
scored.
f
The wild type or the indicated strain was grown on plates containing E. coli expressing dsRNA against the listed gene.
g
Embryos
from parents containing extrachromosomal arrays were scored. Owing to lack of segregation of the arrays, not all of the progeny will inherit
the transgene [78], so some embryos from parents containing WRM0627dD02 still exhibit the umps-1()) phenotype. Only embryos expres-
sing GFP, and therefore having inherited the UMPS-1::GFP array, were scored for intestinal vacuoles.
h
Of the single mutants ⁄ RNAi exam-
ined in the double mutant analysis, only umps-1()) single mutants result in the formation of vacuoles within intestinal cells.
i
Embryos

scored were the progeny of umps-1()); apt-6()) parents, which exhibit 100% maternal effect lethality.
j
Embryos scored were the progeny
of umps-1()) ⁄ umps-1()); glo-1()) ⁄ glo-1(+) parents. The umps-1()); glo-1()) embryos were identified by the loss of the birefringent material
phenotype exhibited by glo-1()) embryos [13].
k
Embryos scored were the progeny of umps-1()) ⁄ umps-1()); pgp-2()) ⁄ pgp-2(+) parents.
Twenty-five per cent of the embryos were expected to be umps-1()); pgp-2()). The double mutants were identified by the loss or reduction
in the amount of birefringent material exhibited by pgp-2()) homozygotes.
l
Embryos scored were the progeny of umps-1()) ⁄ umps-1()); wht-
2()) ⁄ wht-2(+) parents. Twenty-five per cent of the embryos were expected to be umps-1()); wht-2()).
m
pyr-1(cu8) embryos exhibited reces-
sive maternal effect suppression of umps-1(zu456).
n
The strain was scored when grown on plates expressing F33E2.4-derived dsRNA.
F33E2.4 is not required for proper gut granule formation or morphology.
W
A
B
C
Fig. 2. zu456 disrupts the activity of the
bifunctional enzyme UMPS-1, which func-
tions in de novo pyrimidine biosynthesis.
(A) The C. elegans UMPS-1 protein contains
distinct domains that mediate its OPRT and
ODC activities. (B) zu456 alters the pre-
dicted translation initiation site of umps-1
(underlined in bold); use of the next poten-

tial downstream start codon results in the
formation of a short out-of-frame peptide.
(C) The pathway of de novo pyrimidine bio-
synthesis in C. elegans. The proteins that
catalyze each reaction are listed beside the
arrows.
UMPS-1 and gut granule size S. Levitte et al.
1424 FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS
wild-type embryos (n = 19) did so. Thirty-five per cent
(n = 49) of umps-1(zu456) embryos arrested at vari-
ous stages between the bean stage and four-fold stage
of elongation: 10% arrested at the bean stage, 8%
arrested between the 1.5-fold stage and two-fold stage,
12% arrested between the two-fold stage and three-
fold stage, and 5% arrested between the three-fold
stage and four-fold stage. Interestingly, we found that
30% (n = 49) of umps-1(zu456) embryos lysed, typi-
cally prior to elongation. Lysis probably results from
umps-1()) embryos being sensitive to the mechanical
pressure associated with placing embryos between a
3% agarose pad and a coverslip. These observations
indicate that umps-1()) activity is important for
embryonic and larval development, and the arrest and
lysis phenotypes suggest that umps-1(zu456) compro-
mises morphogenesis and the mechanical stability of
the embryo.
The first three enzymatic activities responsible for
de novo pyrimidine biosynthesis in C. elegans are
encoded by pyr-1 [29]. The pyr-1()) mutants, like
umps-1()) mutants, exhibit partially penetrant embry-

onic lethality [31] (Table S2). The lethality of pyr-
1(cu8) is partially suppressed by the addition of uracil
[29], which can be converted into UMP via a salvage
pathway [30]. Similarly, umps-1(zu456) viability was
substantially improved by the addition of uracil to the
growth medium (Table S3). Some of the lethality seen
in pyr-1()) mutants results from a pharyngeal mor-
phogenesis defect that leads to a pharynx-unattached
(Pun) phenotype. The Pun phenotype is probably due
to loss of de novo formation of UMP that is utilized in
proteoglycan synthesis, which is known to be essential
for pharyngeal organogenesis [29]. Like pyr-1())
embryos, umps-1()) embryos exhibited a partially
penetrant Pun phenotype (Table S1). The phenotypic
similarities between umps-1()) and pyr-1()) mutants,
together with the recent observation that umps-1(+)
activity is necessary for 5-fluorouracil-mediated toxicity
in C. elegans [27], a process known to require a func-
tional pyrimidine biosynthesis pathway [31], and the
in vitro biochemical characterization of UMPS-1 [31],
indicate that C. elegans UMPS-1 functions in de novo
pyrimidine biosynthesis.
Embryonic gut granules are enlarged and not
properly formed in umps-1(
)
) embryos
We investigated whether the vacuoles present in
umps-1()) embryos were enlarged gut granules. The
umps-1()) vacuoles contained birefringent material,
and PGP-2 was localized to enlarged compartments in

umps-1()) embryos, suggesting that they were gut
granules (Fig. 1J–L). The integral membrane gut
granule-associated proteins PGP-2::green fluorescent
protein (GFP) (data not shown) [15] and CDF-2::GFP
[32] localized to the limiting membrane of the vacu-
oles in umps-1()) embryos (Fig. 3O,P). Comparison
of PGP-2-stained compartments in wild-type and
umps-1()) pretzel-stage embryos showed average
diameters of 0.41 ± 0.02 lm(n = 60) and 2.6 ±
0.05 lm(n = 50), respectively (± standard error of
the mean). This represents a more than 250-fold
increase in organelle volume in umps-1()) embryos.
If the vacuoles in umps-1()) embryos are gut gran-
ules, then their formation should depend on genes
involved in the formation of gut granules. Mutations
disrupting the functions of the Rab GTPase GLO-1
[13], the AP-3 complex subunit APT-6 [13] and the
ABC transporter PGP-2 [15] result in a Glo (gut gran-
ule loss) phenotype. We constructed umps-
1()); glo()) double mutant embryos, and examined
their intestinal cells for vacuoles. The umps-1());
glo-1()) embryos completely lacked vacuoles, and
umps-1()); apt-6()) embryos typically lacked vacu-
oles (Table 1; Fig. 4D,E). The umps-1( )); pgp-2())
embryos exhibited small vacuoles containing birefrin-
gent material (Table 1; Fig. 4F), consistent with
the partial defect in gut granule biogenesis seen in
pgp-2()) embryos [15]. We conclude that gut granules
are enlarged in umps-1(zu456) embryos.
The umps-1()) mutation affects the characteristics

as well as the size of gut granules. Many gut granules
in umps-1()) embryos did not stain with Lysosensor
Green DND-189 (Fig. 3H), and none of them were
stained by acridine orange (Fig. 3D). Both of these
markers of acidification accumulate in wild-type
gut granules (Fig. 3B,F). VHA-17, a subunit of the
V-ATPase V
0
domain [34], is present on gut granules
and the apical surfaces of wild-type intestinal cells
(Fig. 3R). Although the apical localization was not
altered, VHA-17-labeled compartments similar to those
seen in wild type were lacking in umps-1()) embryos
(Fig. 5T). Detectable levels of VHA-17 were not asso-
ciated with structures resembling enlarged gut granules
(Fig. 5T), consistent with the observed defects in gut
granule acidification in umps-1()) embryos. Unlike
those in wild-type embryos (Fig. 3J), gut granules in
umps-1()) embryos did not stain with Nile Red
(Fig. 3L). These data demonstrate that the properties
of gut granules are dramatically altered in umps-1())
embryos. At present, it is not clear whether this results
from a defect in trafficking of material to the gut gran-
ule or from a dilution of gut granule constituents due
to the dramatic enlargement of gut granule volume
and surface area in umps-1()) embryos.
S. Levitte et al. UMPS-1 and gut granule size
FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS 1425
We tested whether the sizes of other endolysosomal
compartments were as dramatically altered as those of

gut granules in umps-1(zu456) embryos. The morphol-
ogy of early endosomal-associated RAB-5::GFP [13]
and late endosomal-associated RAB-7::GFP [19] was
similar in umps-1(zu456) and wild-type embryos
(Fig. 5B,D,F,H). RAB-5::GFP, RAB-7::GFP, and
the late endosome ⁄ lysosome-associated LMP-1::GFP
proteins, which do not normally associate with gut
granules [15,33], were not obviously enriched on
the limiting membrane of umps-1()) vacuoles
(Fig. 5C,D,G,H,L). Compartments containing LMP-
1::GFP [19] were slightly enlarged in umps-1())
embryos (Fig. 5J,L). Additionally, LMP-1::GFP com-
partments in umps-1()) 1.5-fold stage embryos were
dispersed throughout the cytoplasm, and did not clus-
ter near the apical surfaces of polarized intestinal cells,
as seen in wild-type embryos (Fig. S3). It is possible
that the altered cytoplasmic distribution of LMP-
1::GFP-containing organelles is a consequence of
extremely enlarged gut granules in umps-1())
embryos. LMP-1::GFP is localized to lysosomal com-
partments in C. elegans phagocytic cells and coelomo-
cytes [35,36]. In C. elegans embryonic intestinal cells,
A
B
C
D
E
F
G
H

I
J
K
L
M
N
O
Q
P
R
S
T
Wild type umps-1 (zu456)
DIC Fluorescence DIC Fluorescence
DAPI
Fluorescence
DAPI
Fluorescence
Acridine orangeLysosensorNile RedCDF-2::GFPVHA-17
Fig. 3. Gut granules are enlarged and their properties are altered in umps-1()) pretzel-stage embryos. In wild-type embryos, gut granules
were acidified, being stained by acridine orange (B) and Lysosensor Green (F), contained lipid stained by Nile Red (J), and contained the inte-
gral membrane proteins CDF-2::GFP (N) and VHA-17 (R) (gut granules are marked by white arrows in each panel). The vacuoles within
umps-1()) embryos did not accumulate acridine orange (D) or Nile Red (L); however, some vacuoles accumulated Lysosensor Green [white
arrows in (H)]. The umps-1(zu456) embryos contained greatly enlarged gut granules marked with CDF-2::GFP [white arrows in (P)] and lacked
VHA-17-stained compartments within intestinal cells (T). The apical localization of VHA-17 was present in both wild-type and umps-1(zu456)
embryos [black arrows in (R) and (T)]. The intestine lies between the black arrowheads in all panels. DAPI, 4¢,6-diamidino-2-phenylindole.
UMPS-1 and gut granule size S. Levitte et al.
1426 FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS
we found that mCherry-tagged CPR-6 and F11E6.1
hydrolases were associated with LMP-1::GFP-contain-

ing organelles (Fig. S3). The cpr-6 gene encodes a
cathepsin B protease, and F11E6.1 encodes a glucosyl-
ceramidase, orthologs of which are found in mamma-
lian conventional lysosomes [37]. In umps-1(zu456)
A
B
C
D
E
F
Fig. 4. Suppression of umps-1()) vacuole
formation. DIC microscopy was used to ana-
lyze embryos for intestinal vacuoles, which
are prominent in umps-1()) embryos [white
arrows in (A)]. The umps-1()); pyr-1()) (B)
and umps-1()); wht-2()) (C) embryos lacked
vacuoles and elongated normally. The
umps-1()); apt-6()) (D) and umps-1());
glo-1(zu437) (E) embryos lacked vacuoles
and did not elongate beyond the 1.25-fold
stage. The umps-1()); pgp-2()) embryos
contained small vacuoles [white arrow in (F)]
and arrested elongation prior to the 1.5-fold
stage. The umps-1()) embryos display
vacuoles from the bean stage through
embryogenesis (Fig. S2). White arrowheads
(A, B) flank the pharynx of an embryo
exhibiting the Pun phenotype. Black arrow-
heads flank the intestine in all panels.
Wild type

umps-1 (zu456)
A
B
C
D
E
F
I
J
K
L
G
H
Fig. 5. Analysis of endosomal compartments in umps-1()) embryos. The size and morphology of RAB-5::GFP-labeled endosomes [white
arrows in (B) and (D)] and RAB-7::GFP-labeled endosomes [white arrows in (F) and (H)] were similar in wild-type and umps-1()) pretzel-stage
embryos. LMP-1::GFP-containing compartments were slightly enlarged in umps-1()) embryos [compare white arrows in (J) and (L)]. Black
arrowheads flank the intestine in all panels.
S. Levitte et al. UMPS-1 and gut granule size
FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS 1427
embryos, both proteins were localized to LMP-
1::GFP-labeled compartments, suggesting that these
organelles are properly formed in umps-1(zu456)
embryos (Fig. S3). Thus, umps-1()) appears to most
dramatically affect the formation and morphology of
gut granules.
A role for the ABC transporter WHT-2 in
umps-1(
)
) gut granule enlargement
Lysosomal compartments are highly sensitive to osmo-

tic stress, showing rapid vacuolization on the accumu-
lation of osmotically active material within the
lysosomal lumen [38,39]. Therefore, material within the
gut granule could have a significant impact on its size.
Gut granules contain birefringent material [13,14], cur-
rently of unknown composition [33]. As the birefrin-
gent material is probably present at a high
concentration within the gut granule, we examined its
role in the vacuolization of umps-1()) gut granules.
Disrupting the function of the ABC transporters
MRP-4 and WHT-2 delays the appearance of birefrin-
gent material within gut granules, but does not other-
wise obviously disrupt gut granule biogenesis [33]
(data not shown). The mrp-4()); umps-1()) double
mutant embryos displayed normal-sized vacuoles,
many of which lacked birefringent material, indicating
that the formation of birefringent granules per se is
not required for gut granule enlargement (Table 1).
We used both wht-2(RNAi) and a wht-2 deletion
allele, wht-2(ok2775), to disrupt wht-2(+) activity. In
all of the wht-2()); umps-1()) double mutant combi-
nations, we examined whether there was a loss of, or a
significant reduction in, the number of vacuoles within
intestinal cells (Fig. 4C; Table 1). The wht-2(ok2775)
allele also partially suppressed the embryonic lethality
of umps-1(zu456) (Table S2). Anti-PGP-2 staining
showed that gut granule size was reduced from
an average diameter of 2.6 ± 0.05 lm(n = 50) in
umps-1(zu456) embryos to 0.66 ± 0.04 lm(n = 51)
in umps-1(zu456); wht-2(ok2775) double mutants

(Fig. 6E). In addition, the gut granules in umps-
1(zu456); wht-2(ok2775) embryos were stained by
acridine orange (data not shown). Forty other ABC
transporter mutants were unable to suppress the for-
mation of vacuoles in umps-1(RNAi) embryos
(Table S4). These results indicate that wht-2(+) activ-
ity is necessary for the enlargement of gut granules in
umps-1()) embryos. The lack of similar suppression
by mrp-4()) suggests that wht-2()) mediates this
effect through processes independent of the accumula-
tion of birefringent material within gut granules.
We noticed that many pretzel-stage umps-1());
wht-2()) double mutant embryos exhibited a Pun
phenotype. Nearly 50% of umps-1(zu456); wht-2
(ok2775) and
umps-1(zu456); wht-2(RNAi) embryos
exhibited a Pun phenotype, whereas wht-2( ) ) embryos
did not, and only 7% of umps-1(zu456) embryos did
(Table S1). We investigated whether the genetic inter-
action leading to the Pun phenotype was between
wht-2()) and the de novo pyrimidine biosynthetic
pathway or was specific to umps-1()). pyr-1(cu8);
wht-2(RNAi) embryos did not exhibit a Pun pheno-
type (Table S1), indicating that the Pun phenotype of
umps-1()); wht-2()) represents a specific genetic
interaction between these two genes. The genetic inter-
actions between umps-1()) and wht-2()) implicate the
WHT-2 ABC transporter in the trafficking of metabo-
lites that accumulate in umps-1()) embryos, which
ultimately impinge upon gut granule size and pharyn-

geal morphogenesis.
Analysis of UMPS-1 expression, localization, and
function
To investigate where UMPS-1 functions and how it
might directly regulate gut granule morphology, we
expressed a umps-1::gfp gene under control of the
2.7 kb umps-1 promoter. The UMPS-1::GFP fusion
rescued the Vac phenotype of umps-1(zu456) embryos
(Table 1). UMPS-1::GFP expression was first detected
in early pretzel-stage embryos, where it was expressed
in the intestine and in a few cells in the head and tail
of the animal (Fig. 7A,B). In larval (not shown) and
adult stages, UMPS-1::GFP was expressed in the intes-
tine and neuronal cells located near the nerve ring and
rectum (Fig. 7E,F), which is similar to what has been
documented for an umps-1 promoter-driven reporter
[27].
The umps-1(zu456) embryos displayed a strict,
maternal effect Vac phenotype (Table 1). This could
result from metabolic processes involving UMPS-1 at
work in the adult intestine that impact on embryonic
gut granules. For example, yolk proteins derived from
the adult intestine are transferred into oocytes, where
they accumulate in the embryonic intestine [40,41].
We performed RNAi on rde-1()); elt-2p::rde-1(+)
animals, which are only susceptible to feeding
based RNAi in larval and adult intestinal cells [42].
We found that none of the embryos exhibited a
Vac phenotype (Table 1), suggesting that inhibiting
umps-1(+) in the adult intestine does not impact on

embryonic gut granule size.
We next considered whether the loss of umps-1
expression in the germline leads to enlarged gut
UMPS-1 and gut granule size S. Levitte et al.
1428 FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS
granules. We analyzed the effects of umps-1(RNAi)
on rrf-1(pk1417) animals, which are defective for
somatic RNAi but are competent for germline RNAi
[43]. We found that rrf-1(pk1417) animals were as
sensitive to umps-1(RNAi) as wild-type animals
(Table 1). Thus, umps-1 expression in the germline is
necessary to prevent the enlargement of embryonic
gut granules, suggesting that the maternal effect Vac
phenotype of umps-1()) probably results from the
maternal contribution of umps-1(+) to embryonic
progeny. This could take the form of UMPS-1
protein, umps-1 mRNA, and ⁄ or UMPS-1 metabolic
activity in the germline.
Mammalian UMPS is localized to the cytoplasm
[44,45], and C. elegans UMPS-1 does not contain any
obvious organelle targeting or retention motifs, sug-
gesting a similar localization. In embryonic intestinal
cells, UMPS-1::GFP was distributed throughout the
cytoplasm, without any obvious organelle association
(Fig. 7A,B). However, we often observed UMPS-
1::GFP near the apical surface of the embryonic
intestine (Fig. 7A,B). In adult intestinal cells, UMPS-
1::GFP appeared to be uniformly localized throughout
the cytoplasm (Fig. 7C,D). These data suggest that
UMPS-1 is a cytoplasmic protein that is not associated

with the gut granule.
Accumulation of orotic acid probably leads to
enlarged gut granules in umps-1(
)
) embryos
Mutations that disrupt the function of human UMPS
result in orotic aciduria, a disease characterized by
megaloblastic anemia, failure to thrive, and urinary
excretion of large amounts of orotic acid [6,30,46].
Disrupting the function of the Drosophila UMPS-
encoding gene rudimentary-like results in sterility,
reduced viability, wing and leg morphological defects,
and accumulation of orotic acid [47–49].
Many of the phenotypes resulting from loss of
UMPS activity are due to pyrimidine auxotrophy
[30,47]. We therefore considered the possibility that
A
B
C
D
E
Fig. 6. Suppression of enlarged gut granules in umps-1())
embryos. Embryos lacking umps-1(+) activity had enlarged gut
granules marked with antibodies against PGP-2 [white arrows in
(B)]. The pyr-1()) embryos had gut granules that were slightly
enlarged [white arrows in (C)] in comparison with wild-type
embryos [white arrow in (A)]. The gut granules of umps-1());
pyr-1()) and umps-1()); wht-2()) embryos were dramatically
reduced in size [white arrows in (D) and (E)], and were similar in
size to gut granules in pyr-1()) embryos [white arrows in (C)]. The

intestine of pretzel-stage embryos is flanked by black arrowheads
in all panels.
S. Levitte et al. UMPS-1 and gut granule size
FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS 1429
reductions in the levels of UMP, the end-product of
the de novo pyrimidine biosynthetic pathway, result in
gut granule enlargement. The pyr-1()) and umps-1())
embryos should have a similar reduction in UMP lev-
els; however, pyr-1(cu8) and pyr-1(RNAi) did not
result in vac embryos (Table 1). Interestingly, PGP-2-
labeled gut granules were slightly enlarged in pyr-1())
embryos (Fig. 6C), from an average diameter of
0.41 ± 0.02 lm(n = 60) in wild-type embryos to
0.67 ± 0.04 lm(n = 40) in pyr-1(cu8) embryos, sug-
gesting that the reduction in UMP levels has a minor
effect on gut granule size. Addition of 5 mgÆmL
)1
ura-
cil to the growth medium significantly improved the
viability of umps-1(zu456) embryos (Table S3), proba-
bly owing to the synthesis of UMP via a salvage path-
way; however, it did not suppress the Vac phenotype
of umps-1()) (Table 1). Therefore, the reduction in
UMP levels resulting from umps-1()) appears to play
only a minor role in promoting gut granule enlarge-
ment.
The loss of UMPS activity, in a number of different
species, results in the accumulation of orotic acid
[30,47]. We investigated whether the accumulation of
orotic acid in umps-1()) mutants leads to gut granule

enlargement. As PYR-1 acts before UMPS-1 in the lin-
ear pathway of UMP synthesis, pyr-1( )) mutants
should lack orotic acid, the substrate of UMPS-1
(Fig. 2C). We found that umps-1()); pyr-1())
embryos completely lacked intestinal vacuoles (Fig. 4B;
Table 1), and that the diameter of PGP-2-labeled
gut granules was decreased in umps-1(zu456);
pyr-1(RNAi) embryos to 0.59 ± 0.03 lm(n = 40)
(Fig. 6D). The umps-1()); pyr-1()) embryos exhibited
gut granules that stained with Nile Red and acridine
orange (Fig. S4), characteristics that are lacking in
umps-1()) embryos (Fig. 3). Moreover, pyr-1(cu8)
partially suppressed the embryonic lethality caused by
umps-1(zu456) (Table S2). The suppression of umps-
1()) phenotypes by removal of pyr-1(+) activity
strongly suggests that the accumulation of orotic acid
in umps-1()) mutants leads to gut granule enlarge-
ment and contributes to embryonic lethality.
Vacuoles in umps-1(
)
) larvae are osmotically
sensitive
Lysosomes can swell and form vacuoles as a result of
the lumenal accumulation of osmotically active com-
pounds [38,50,51]. We considered the possibility that
umps-1()) leads to osmotically induced gut granule
enlargement. In cultured cells, increasing the osmolar-
ity of the growth medium reverses osmotically induced
swelling of lysosomes [52]. To determine whether
enlargement and vacuolization of gut granules in

umps-1(zu456) animals could be reversed by hyper-
tonic conditions, we mounted newly hatched L1-stage
umps-1(zu456) larvae in media with increasing concen-
trations of NaCl. Incubating umps-1(zu456) larvae in
water or 100 mm NaCl did not alter the number or
morphology of vacuoles over 45 min (Fig. 8A,B;
Fig. S5). Strikingly, incubation of umps-1(zu456) lar-
vae in 300 mm NaCl led to a five-fold to 10-fold
reduction in the number of vacuoles within 15 min
(Fig. 8C–E). A similar effect was seen after 30–45 min
when umps-1(zu456) larvae were incubated in 200 mm
NaCl (Fig. S5). The morphology of wild-type L1-stage
intestinal cells was not dramatically affected by
exposure to 300 mm NaCl (data not shown). The alter-
ations in vacuole appearance resulted from osmotic
effects, and were not specific to NaCl, as incubation in
A
B
C
D
E
F
20 µ
µ
m
80
µ
m
Fig. 7. Expression and localization of
UMPS-1::GFP. Pretzel-stage (A, B) and

adult-stage (C–F) umps-1(zu456) animals
expressing UMPS-1::GFP are shown.
UMPS-1 was expressed in intestinal cells at
both stages (the intestine is located
between the black arrowheads) (A, B and E,
F), where it is cytoplasmically localized (B,
D). In embryonic intestinal cells, UMPS-
1::GFP was often enriched near the apical
surface [white arrows in (A) and (B)].
UMPS-1::GFP was additionally expressed in
neuronal cells [white arrowheads in (B) and
(F)] and the adult ventral nerve cord [white
arrow in (F)]. The intestinal lumen is marked
with a white arrow in (A)–(D).
UMPS-1 and gut granule size S. Levitte et al.
1430 FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS
484 mm sucrose, which is osmotically equivalent to
300 mm NaCl [53], also reduced the number of vacu-
oles in umps-1(zu456) larvae (data not shown). The
number and morphology of intestinal vacuoles present
within ppk-3(n2668) L1-stage larvae were not reduced
by hypertonic conditions (data not shown), indicating
that exposure to hypertonic medium does not generally
alter vacuole morphology ⁄ appearance within larval
intestinal cells. These data show that increased osmo-
larity can rapidly and substantially reduce the number
of vacuoles in umps-1(zu456) larvae.
Accumulation of orotic acid probably leads to
lethality in umps-1(
)

); glo(
)
) embryos
The glo embryos exhibit a loss of gut granules, but they
develop and elongate to four-fold length normally [13].
The umps-1(zu456) embryos exhibited significant
embryonic lethality (Table S2), characterized by 35%
(n = 49) of embryos arresting at various stages between
the bean and four-fold stage of elongation; however,
none of the embryos arrested between the 1.25-fold and
1.5-fold stages. Thirty-five per cent of umps-1())
embryos fully elongated (four-fold) (n = 49). We
observed early embryonic arrest in three different umps-
1(zu456); glo()) double mutants that was distinct
from what we observed in the umps-1(zu456) mutant
alone. One hundred per cent of umps-1(zu456); glo-
1(zu437) embryos (n = 22) (Fig. 4E) and umps-
1(zu456); pgp-2(kx55) embryos (n = 17) (Fig. 4F)
arrested at the 1.25–1.5-fold stage of elongation. Sixty-
three per cent of umps-1(zu456); apt-6(ok429) embryos
(n = 59) arrested as 1.25–1.5-fold stage embryos
(Fig. 4D). Moreover, we were never able to isolate an
umps-1(zu456); glo()) line from any of the crosses
used to create the double mutant embryos, which is
indicative of a synthetic lethality.
The synthetic lethality could be due to a reduction in
UMP levels or a build-up of orotic acid, both of which
are predicted to result from loss of umps-1(+) function.
We found that the reduction in UMP levels did not
significantly contribute to the arrest phenotype of

umps-1()); glo()) double mutants, as apt-6(ok429);
pyr-1(RNAi) (n = 37) embryos did not arrest, and only
8% of glo-1(zu437); pyr-1(RNAi) (n = 40) and 5% of
pgp-2(kx55); pyr-1(RNAi) (n = 41) embryos arrested
during embryogenesis. If the accumulation of orotic
acid promotes the lethality of umps-1()); glo())
embryos, then pyr-1(RNAi) should suppress the
embryonic arrest phenotype. We tested this possibility
using glo-1(zu437); umps-1(zu456) embryos, which all
arrest during development. We found that 100% of
glo-1(zu437); umps-1(zu456); pyr-1(RNAi) embryos
(n = 40) elongated to four-fold and were viable. In fact,
we were able to generate and maintain a viable
glo-1(zu437); umps-1(zu456) strain, as long as it was
grown on pyr-1(RNAi) feeding plates. These results are
consistent with gut granules providing a protective func-
tion, probably acting to suppress the lethality associated
with increased levels of orotic acid in umps-1())
embryos.
Discussion
Role of the V-ATPase and PPK-3 in the control of
gut granule size
We have shown that, with the exception of ppk-3()),
disrupting the function of known regulators of endolys-
osomal compartment size does not lead to significant
100
90
80
70
60

50
40
30
20
10
0
>50 25 to 50 10 to 24 5 to 9 0 to 4
Number of vacuoles
% of L1s
0 min (n = 20)
15 min (n = 20)
30 min (n = 20)
A
B
C
D
E
Fig. 8. Vacuole number rapidly decreases in umps-1()) larvae exposed to hypertonic media. Newly hatched umps-1(zu456) L1-stage larvae
were mounted in 0 m
M (A, B) or 300 mM NaCl (C, D), and vacuoles (white arrows) were visualized with DIC microscopy. The same animal
is shown at each time point for each treatment (A–D). The number of clearly defined vacuoles present within larvae exposed to 300 m
M
NaCl was scored over 30 min using the same population of animals (E).
S. Levitte et al. UMPS-1 and gut granule size
FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS 1431
enlargement of embryonic gut granules (Table 1; Fig. 1;
Fig. S1). Inhibiting the activity of ppk-3()) and its
mammalian or yeast orthologs [21] results in dramati-
cally enlarged late endolysosomal compartments
[21,23]. It has been reported that C. elegans ppk-3())

adults display greatly enlarged autofluorescent gut gran-
ules [21]. However, the ppk-3()) animals in which this
phenotype was observed overexpressed both LMP-
1::GFP and RAB-7::RFP [21], which could perturb
normal membrane trafficking pathways, impacting on
gut granule morphology. We analyzed the diameter of
autofluorescent gut granules within ppk-3()) embryos
and adults not expressing these transgenes, and found
that gut granules were only slightly enlarged, if at all
(Fig. 1; Fig. S1). As there is strong evidence that the
ppk-3()) alleles that we analyzed significantly or com-
pletely disrupt ppk-3(+) function [21], we propose that
PPK-3 plays only a minor role in regulating gut granule
size. The subtle effects of ppk-3()) on gut granule size,
when compared with the significant enlargement of
other endolysosomal compartments in C. elegans [21],
probably reflect the distinct pathways and mechanisms
involved in the formation ⁄ dynamics of LROs and con-
ventional late endosomes ⁄ lysosomes.
Genetic inhibition of V-ATPase function can lead to
the formation of enlarged endolysosomal compart-
ments [54–56], but its activity has not been implicated
in the control of LRO size. We found that disrupting
the activity of unc-32, which encodes a V-ATPase sub-
unit, led to the dramatic enlargement of embryonic
compartments with gut granule characteristics (Fig. 1).
This observation suggests that the V-ATPase, which is
localized to many LROs [57,58], might have a general
role in controlling LRO morphology. It will be inter-
esting to examine whether endocytic trafficking and ⁄ or

the dynamics of organelle fission–fusion, both of which
can be regulated by the V-ATPase [54,59–61], are
altered in unc-32()) embryos.
Gut granule enlargement in umps-1(
)
) embryos
There are evolutionarily conserved and organelle-spe-
cific regulators of LRO formation and morphology.
Common trafficking pathways have been identified
that mediate the formation of most LROs examined to
date [2]. However, despite LROs having shared mecha-
nisms of biogenesis, their physiological functions are
quite distinct, and stem from the distinctive protein
composition of each type of LRO [1]. LRO-specific
effectors endow each type of organelle with particular
cellular properties, which probably include those
important for regulating LRO biogenesis and morphol-
ogy. For example, ABCA3 appears to be specifically
involved in the formation of lamellar bodies [62,63].
We believe that our studies of UMPS-1 reveal a
specific functional property of the C. elegans gut gran-
ule that is relevant to its morphology.
umps-1()) embryos contained greatly enlarged gut
granules (Figs 1 and 3), whereas the size of other
endolysosomal compartments did not appear to be as
significantly altered (Fig. 5). Why does loss of
umps-1(+) activity lead to gut granule enlargement?
UMPS-1 functions in the de novo synthesis of pyrimi-
dines by catalyzing the formation of UMP [27], which is
used in a variety of cellular processes [64]. Disrupting

another catalyst in the pathway, PYR-1, did not lead to
dramatically enlarged gut granules (Table 1; Fig. 6),
indicating that decreased levels of pyrimidines or their
derivatives do not promote the alterations in gut
granule morphology seen in umps-1()) embryos.
UMPS-1::GFP is a cytoplasmically localized protein
that is not enriched on the gut granule (Fig. 7), and
UMPS-1 does not contain any structural motifs impli-
cated in processes that control organelle trafficking or
biogenesis. We therefore do not think that UMPS-1
directly regulates membrane dynamics to control gut
granule size.
Disrupting UMPS function results in the accumula-
tion of its substrate, orotic acid [30,47], which, as we
show, probably promotes gut granule enlargement.
PYR-1 acts before UMPS-1 in the de novo synthesis of
pyrimidines (Fig. 2), so umps-1()); pyr-1()) embryos
should lack orotic acid. Consistent with the idea that
increased levels of orotic acid underlie the umps-1())
phenotype, pyr-1()); umps-1()) embryos contained
gut granules with wild-type characteristics that were not
substantially enlarged (Table 1; Figs 4 and 6; Fig. S4).
In addition to gut granules being enlarged, their
characteristics were altered in umps-1()) embryos.
Most notably, umps-1()) gut granules were not
stained by acridine orange, were weakly stained by
Lysosensor Green, and did not obviously contain the
V-ATPase subunit VHA-17. These observations indi-
cate that the enlarged gut granules in umps-1())
embryos are not properly acidified. Nile Red also

failed to accumulate in the gut granules of umps-1())
embryos. These phenotypes could result from defects
in gut granule formation, defects in the activity of gut
granule-associated factors, and ⁄ or the dilution of mar-
ker signals caused by the massive increase in gut gran-
ule surface area and volume. In umps-1()) embryos,
PGP-2 and CDF-2::GFP are properly localized to the
gut granule membrane, birefringent material accumu-
lates within the gut granule lumen, and umps-1())
does not cause the elongation arrest phenotype
exhibited by double mutants lacking the activity of
UMPS-1 and gut granule size S. Levitte et al.
1432 FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS
umps-1(+) and known gut granule biogenesis genes,
suggesting that gut granule formation is not generally
compromised by loss of umps-1(+). Disrupting the
activity of unc-32 and other V-ATPase subunits leads
to gut granule enlargement and loss of gut granule
acidification (Fig. 1 and our unpublished observa-
tions). However, loss of V-ATPase activity is associ-
ated with changes in the endolysosomal system that are
not seen in umps-1()) embryos (our unpublished obser-
vations), suggesting that the altered characteristics of
umps-1()) gut granules do not result from defects in
V-ATPase function. The pyr-1()) mutation suppressed
the formation of enlarged gut granules in umps-1())
embryos (Figs 4 and 6), and the gut granules in umps-
1()); pyr-1()) embryos stained with acridine orange
and Nile Red (Fig. S4). Given the biochemical activity of
pyr-1(+), this suppression probably results from block-

ing orotic acid accumulation in umps-1()) embryos.
Although orotic acid could directly interact with, and
disrupt the function of, trafficking factors or the V-AT-
Pase, given the lack of precedent for this model, we
favor the interpretation that gut granule characteristics
are restored in umps-1()); pyr-1()) embryos simply
through the reduction in gut granule size.
Fluorescent dyes that should be unable to cross cel-
lular membranes, when fed to C. elegans adults, accu-
mulate in the gut granule [12]. We examined whether
increased amounts of orotic acid were sufficient for gut
granule enlargement by feeding animals high levels of
orotic acid. These treatments did not promote the for-
mation of intestinal vacuoles at any stage of develop-
ment (data not shown). However, umps-1()) larvae
and adults do not contain enlarged gut granules
(Fig. S2), so increased levels of orotic acid might not
be expected to alter gut granule size at these stages. It
is unclear whether orotic acid fed to adults can be
transported into oocytes, the route by which it would
get into embryos. Orotic acid is an anionic cyclic
carboxylic acid that will not rapidly diffuse across lipid
membranes [65]. Whereas prokaryotic cells have well-
described orotic acid transporters [66,67], it has been
suggested that most eukaryotic cells do not actively
transport orotic acid across the plasma membrane
[65,68,69].
Although there are no known orotic acid transporters
in eukaryotes, the excretion of large amounts of orotic
acid associated with orotic aciduria suggests that there

might be unidentified proteins with this activity. ABC
transporters actively transport a broad spectrum of
organic anions [70], and have been found to prevent the
cytoplasmic accumulation of toxic metabolic intermedi-
ates [71]. We identified WHT-2, an ABCG subfamily
member, as being necessary for the enlargement of gut
granules in umps-1()) embryos (Table 1; Figs 4 and 6).
Activity of wht-2(+) has been shown to impact on gut
granule morphogenesis, as the accumulation of birefrin-
gent material in gut granules is delayed in wht-2())
embryos [33]. Possibly, WHT-2 is localized to the gut
granule, where it mediates the transport of orotic acid
from the cytoplasm to the gut granule.
The swelling and vacuolization of lysosomes has long
been known to result from the lumenal accumulation of
osmotically active compounds [38,50,51]. If orotic acid
was actively transported into the gut granule, it could
induce osmotic stress and organelle enlargement. The
size of lysosomes that have become enlarged because of
osmotic swelling can be reduced by exposure to hyper-
tonic medium [52]. Consistent with orotic acid accumu-
lation promoting osmotic enlargement of gut granules,
we found that the number of vacuoles in umps-1()) lar-
vae rapidly decreased when they were exposed to hyper-
tonic conditions (Fig. 8; Fig. S5).
We propose that the formation of grossly enlarged
gut granules in umps-1()) embryos compromises
embryonic viability. Reduced levels of pyrimidines
resulting from defects in de novo synthesis cause lethal-
ity, which is, in part, associated with abnormal pha-

ryngeal morphogenesis [29]. However, our analysis of
embryonic and larval phenotypes strongly suggests
that loss of umps-1(+) activity is associated with
more severe lethality than can be attributed to the loss
of pyrimidines synthesized by the de novo pathway
(Table S2). Importantly, both pyr-1()) and wht-2()),
which suppress gut granule enlargement in umps-1())
embryos (Figs 4 and 6), significantly enhance the sur-
vival of umps-1()) embryos (Table S2). We have
found that the embryonic lethality caused by umps-
1()) is associated with arrest during morphogenesis
and mechanical integrity. It is therefore possible that
enlarged gut granules present within intestinal cells
interfere with the force-generating mechanisms that
elongate the embryo and provide resistance to rupture,
processes that appear to be linked at the stages of
embryogenesis affected by umps-1()) [72].
Mutations disrupting gut granule formation caused
synthetic lethality in combination with umps-1()). The
lethality is probably due to the accumulation of orotic
acid, and not to defects in de novo pyrimidine biosyn-
thesis, as pyr-1()) did not similarly cause synthetic
lethality with gut granule biogenesis mutants, and
instead suppressed the lethality of umps-1());
glo-1()) (Table 1). Accumulation of orotic acid leads
to phenotypes in Drosophila and human cells that are
consistent with cytotoxic effects [30,47]. One function
of the gut granule, which would also explain why it is
specifically affected in umps-1()) embryos, might be
S. Levitte et al. UMPS-1 and gut granule size

FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS 1433
to mitigate these effects by actively taking up and stor-
ing orotic acid.
Irrespective of the mechanism by which umps-1())
promotes gut granule enlargement, it will be interesting
to examine the molecular and cellular processes that
facilitate the specific and dramatic increase in gut gran-
ule membrane and volume exhibited by this mutant.
Experimental procedures
C. elegans strains and genetic manipulations
Nematodes were grown at 22 °C, as described previously
[73], on NGM medium supplemented with 2 lgÆmL
)1
uracil
to facilitate the growth of the Escherichia coli uracil auxo-
troph OP50. OP50 provides a dietary source of pyrimidines
that can be converted to UMP via salvage pathways. N2
was used as the wild-type strain. References for the follow-
ing alleles used in this study can be found at http://
wormbase.org: alx-1(gk275), apt-6(ok429), glo-1(zu437),
mrp-4(ok1095), pgp-2(kx55), ppk-3(n2668), ppk-3(ok1150),
ppk-3(zu443), rab-10(dx2), rde-1(ne219), rme-1(b1045),
rrf-1(pk1471), rrf-3(pk1426), tat-1(kr15), umps-1(zu456),
unc-32(f121), unc-32(f123), unc-32(kx45), unc-32(zu436),
unc-32(zu438), unc-32(zu440), unc-32(zu445), pyr-1(cu8),
and wht-2(ok2775). Animals containing the following
transgenes were used: amIs4[cdf-2::gfp; unc-119(+)] [32],
mIs11[myo-2::gfp], OLB11 dusIs [elt-2
p
::rde-1(+)] [42],

pwIs50[lmp-1::gfp; unc-119(+)] [35], pwIs72[vha-6
p
::rab-
5::gfp; unc-119(+)] [13], pwIs170[vha-6
p
::rab-7::gfp; unc-
119(+)] [19], kxEx98[pgp-2::gfp; rol-6
D
] [15], kxEx141
[cpr-6::mCherry; rol-6(su1006)
D
] (this work), kxEx148
[F11E6.1::mCherry; rol-6(su1006)
D
] (this work), and
kxEx167[umps-1::gfp; rol-6(su1006)
D
] (this work). RNAi
was performed as previously described [74], using bacterial
clones from a C. elegans RNAi feeding library (Geneservice,
Cambridge, UK).
The ppk-3(zu443), unc-32(kx45), unc-32(zu436), unc-32
(zu438), unc-32(zu440), unc-32(zu445) and umps-1(zu456)
alleles were identified in maternal and zygotic effect screens
for mutants with an embryonic Vac phenotype. The mutants
were isolated following mutagenesis with EMS as previously
described [75]. The zu443 allele mapped to the X chromo-
some, and ppk-3(n2668) ⁄ zu443 L4-stage animals derived
from mating ppk-3(n2668) males with zu443 hermaphrodites
exhibited the ppk-3()) vacuole phenotype [21], indicating

that zu443 is an allele of ppk-3. The ppk-3(zu443) line was
backcrossed with N2 prior to analysis. Zygotic lethal Vac
mutants stood out as carrying unc-32 mutations [25]. Alleles
of unc-32 were identified by mating animals heterozygous for
the vac allele with unc-32(f123) ⁄ + males marked with a
GFP-expressing chromosome. Approximately one-eighth of
the outcross progeny, recognized by GFP expression, exhib-
ited the intestinal Vac phenotype.
The umps-1(zu456) line was backcrossed with the wild type
six times prior to our genetic and phenotypic analyses. The
umps-1(zu456) line was shown to exhibit a strict maternal
effect Vac phenotype, using standard genetic techniques
(Table 1). To generate umps-1 double mutants, umps-1
(zu456) males were mated with pyr-1()) or glo() ) strains.
Vac F
2
progeny of these crosses, which were heterozygous for
the pyr-1()) or glo()) phenotype, were isolated. At least two
independently isolated lines were scored for each cross.
Microscopy
Microscopic analysis was carried out using a Zeiss Axio-
skop II Plus microscope (Thornwood, NY, USA), config-
ured for DIC, polarization, and fluorescence imaging. An
Insight Spot QE 4.1 digital camera controlled with spot
basic software (Diagnostic Instruments, Sterling Heights,
MI, USA) was used for image capture. Adults and larvae
were mounted in 1 · M9+10mm levamisole (Sigma, St
Louis, MI, USA) and immobilized prior to imaging.
Pretzel-stage embryos were immobilized by hypoxia prior
to imaging by mounting embryos with excess living E. coli.

Vacuoles within intestinal cells were recognized by DIC
microscopy as obvious clearings, with a uniform interior,
not present within the wild type. The autofluorescent mate-
rial within adult gut granules [12] was visualized with a
Zeiss Ex:BP480 ⁄ 20 Em:BP530 ⁄ 20 filter set. Acidic intestinal
compartments were stained with acridine orange (Sigma) as
previously described [13], and visualized with a Zeiss 15
Ex:BP586 ⁄ 12 Em:LP590 filter. Acidified compartments were
also identified by examining the embryonic progeny of ani-
mals grown on seeded NGM plates containing 2 lm Lysosen-
sor Green DND-189 (Invitrogen, Carlsbad, CA, USA),
and scored with a Zeiss Ex:BP480 ⁄ 20 Em:BP530 ⁄ 20 filter
set. Nile Red staining of adults and embryos was per-
formed as previously described [15]. GFP-tagged and
mCherry fluorescently tagged proteins were visualized in
living embryos using Zeiss Ex:BP480 ⁄ 20 Em:BP530 ⁄ 20 and
Zeiss 31 Ex:BP565 ⁄ 30 Em:BP620 ⁄ 60 filters, respectively.
Polarization microscopy was used to detect the birefringent
material present within embryonic gut granules [13]. The
colocalization of vacuoles and fluorescent dyes ⁄ proteins
was analyzed by manually assessing DIC and fluorescence
images merged using photoshop cs2 (Adobe, San Jose,
CA, USA). To determine the size of adult gut granules,
images of fluorescein isothiocyanate gut granule-associated
autofluorescence within intestinal cells of adults were
captured. To determine the size of embryonic gut granules,
images of anti-PGP-2-stained 1.5-fold or pretzel-stage
embryos were captured. Gut granule diameter was mea-
sured using imagej 1.42 software ( />Gut granules were measured in at least three animals of
each genotype. Embryos were stained with anti-RAB-5

[76], anti-PGP-2 [15], anti-VHA-17 ⁄ FUS-1 [34] and
UMPS-1 and gut granule size S. Levitte et al.
1434 FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS
anti-GFP (3E6; Qbiogene, Carlsbad, CA, USA) sera, as
previously described [77].
Embryonic development was examined by placing two-
cell or bean-stage embryos on freshly prepared 3% agarose
pads. Excess bacteria were removed by rinsing with a
mouth pipette, and a coverslip was gently placed on top of
the embryos. Embryos of the 1.5–2-fold stage placed near
these embryos were monitored for movement to ensure that
embryogenesis was not halted by hypoxia. Embryos were
scored every 2 h by DIC microscopy, and were judged to
be arrested if they remained at the same stage of develop-
ment ⁄ morphogenesis for two consecutive time points.
Between scoring, the embryos were kept in a humid cham-
ber at 22 °C.
To assess the effects of osmolarity upon vacuolated gut
granules, adults and larvae were rinsed from NGM plates,
leaving the embryos. Six to eight hours later, newly
hatched L1-stage larvae were picked into drops of
0–300 mm NaCl on freshly prepared 3% agar pads made
with 0–300 mm NaCl. A coverslip was placed over the lar-
vae, and the number of vacuoles was scored at 15 min
intervals. Slides were kept at 22 °C in a humid chamber
between time points.
Cloning umps-1
Three-factor genetic mapping with unc-69 dpy-18⁄ zu456
placed zu456 very close to unc-69. Four-factor genetic
mapping with sma-2 ced-7 unc-49 ⁄ zu456 placed zu456 in

the ced-7 unc-49 interval, near unc-49 (mapping data
available upon request). An RNAi screen of genes in this
region identified a single clone, JA:T07C4.1, as having an
embryonic Vac phenotype indistinguishable from zu456
embryos. We sequenced the region between the T7
sequences of this clone, and confirmed that it contained
sequences targeting T07C4.1. A large number of
T07C4.1-derived cDNAs have been identified and
sequenced, leading to a well-supported model of mRNA
splicing for this gene, strongly suggesting that T07C4.1 is
not alternatively spliced and that all of the predicted ex-
ons are present within the mature mRNA (Worm-
base WS200). Two of two stably transmitting umps-
1(zu456) lines injected with fosmid wrm0627Dd02 at
10 ngÆlL
)1
and the marker pRF4[Rol-6
D
] [78] at
100 ngÆlL
)1
showed rescue of the Vac phenotype. Phu-
sion high-fidelity DNA polymerase (New England Biol-
abs, Beverly, MA, USA) was used to amplify the umps-1
predicted coding sequence from zu456. The resulting
DNA products were sequenced (OHSU Core Facility) to
identify the zu456 molecular lesion.
Generation of fusion constructs
The cpr-6::mCherry and F11E6.1::mCherry translational
fusions designed to produce C-terminal mCherry-tagged

proteins under control of their own promoters were
constructed using PCR fusion [79]. The genomic region
containing 2700 bp of sequence upstream of the predicted
start codon of cpr-6 and the entire predicted coding cpr-6
sequence was amplified with Phusion polymerase from N2
genomic DNA using P593 5¢-AGGTAGGACACTTTTTG
CTACGCAACC-3¢ and P595 5¢-TATCTTCTTCACCCT
TTGAGACCATGTAGTTGTCATCGTAGACGTGGC-3¢.
The genomic region containing 1400 bp of sequence
upstream of the predicted start codon of F11E6.1 and
the entire predicted coding sequence predicted to include
the 3¢-end of splice forms F11E6.1a and F11E6.1b (Worm-
base WS200) was amplified with Phusion polymerase from
N2 genomic DNA using P580 5¢-TCTGTTTCCGATCTTA
CTTGAGCGTTG-3¢ and P582 5¢-TATCTTCTTCACC
CTTTGAGACCATTTTTCTTTTCTTCCAAATCACTG-3¢.
The mCherry coding sequence and flanking let-858 3¢-UTR
were amplified from a pvha-6p::GTWY::mCherry plasmid
[80] using P568 5¢-ATGGTCTCAAAGGGTGAAGAAGA
TAAC-3¢ and P570 5¢-GGATTCTAGACTAGTTTTCCTT
CCTCC-3¢. The cpr-6 and mCherry PCR products were
fused using P612 5¢-TGGACCGTTCTCAGAAAGTAA
CTCCGC-3¢ and P598 5¢-TTTATGTTTTCTTTTAAAC
CTTCCTCC-3¢. The resulting cpr-6::mCherry product was
coinjected into N2 at 5 ngÆlL
)1
with pRF4[Rol-6
D
] at 100
ngÆlL

)1
. kxEx141[cpr-6::mCherry; rol-6(su1006)
D
] was
used in the analysis presented here. The F11E6.1 and
mCherry PCR products were fused using P581 5¢-TCA
AAATTGGTGTGAATGACGAGAAAC-3¢ and P598. The
resulting F11E6.1::mCherry product was coinjected into N2
at 20 ngÆlL
)1
with pRF4[Rol-6
D
] at 100 ng ÆlL
)1
. kxEx148
[F11E6.1::mCherry; rol-6(su1006)
D
] was used in the ana-
lysis presented here.
The rescuing translational reporter umps-1::gfp made
using the umps-1 genomic sequence was constructed using
PCR fusion [79]. The genomic region containing 2700 bp of
sequence upstream of the predicted start codon of umps-1
and the entire coding sequence was amplified with Phusion
polymerase from N2 genomic DNA using P616 5¢-GA
ACTGGCAAATTTTCGAACCTGTCTG-3¢ and P618
5¢-AGTCGACCTGCAGGCATGCAAGCTAATGCTATC
GTCGCTTCTCGTGAG-3¢. The gfp coding sequence and
flanking unc-54 3¢-UTR were amplified from pPD95.75
(Addgene, Cambridge, MA, USA) using P399 5¢-AGCTT

GCATGCCTGCAGGTCGACT-3¢ and P266 5¢-AAGG
GCCCGTACGGCCGACTAGTAGG-3¢. The two PCR
products were fused using P617 5¢-CAGTGTCCTATA
ATTTAAACGCGACTG-3¢ and P267 5¢-GGAAACAGT
TATGTTTGGTATATTGGG-3¢. The resulting umps-
1
p
::umps-1::gfp product was coinjected into umps-1(zu456)
at 18 ngÆlL
)1
with pRF4[Rol-6
D
] at 100 ngÆlL
)1
. umps-
1(zu456); kxEx167[umps-1::gfp; rol-6(su1006)
D
] was used
in the analysis presented here, and two of two other inde-
pendently derived lines showed a similar expression pattern
S. Levitte et al. UMPS-1 and gut granule size
FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS 1435
and subcellular distribution of GFP. Three of three lines
rescued the umps-1()) Vac phenotype.
Acknowledgements
We are especially grateful to J. Priess, in whose labora-
tory G. Hermann identified most of the vac mutants.
We thank R. Fitch, T. Schaetzel-Hill and other stu-
dents in BIO361 at Lewis & Clark College during 2008
for sharing their analysis of gut granule enlargement in

ppk-3()) embryos. We are grateful to O. Bossinger,
A. Chisholm, J. Fares, B. Grant, K. Kornfeld,
J. Laporte, the Caenorhabditis Genetics Center, the
C. elegans Knockout Consortium and the National
Bioresource Project for C. elegans for strains and plas-
mids. This work was supported by grants from the
Beckman Foundation, the John S. Rogers Summer
Research Program, and the National Science Founda-
tion (MCB-0716280).
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Supporting information
The following supplementary material is available:
Fig. S1. Gut granules are not dramatically enlarged in
ppk-3()) embryos and adults.
Fig. S2. The umps-1()) animals display vacuoles
between the bean and L1 stages of development.
Fig. S3. CPR-6::mCherry and F11E6.1::mCherry are
properly localized to LMP-1::GFP compartments in
umps-1(zu456) embryos.
Fig. S4. Presence of acridine orange-stained and Nile
Red-stained compartments in umps-1()); pyr-1())
embryos.
Fig. S5. Effects of increased osmolarity on vacuoles
within umps-1(zu456) embryos.
Table S1. Analysis of Pun phenotypes.
Table S2. Analysis of embryonic and larval lethality.
Table S3. Suppression of umps-1(zu456) lethality by
the addition of uracil.
Table S4. Screening ABC transporters for suppression
of vacuole formation in umps-1()) embryonic intesti-
nal cells.
This supplementary material can be found in the
online online version article.
Please note: As a service to our authors and readers,
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from supporting information (other than missing files)
should be addressed to the authors.
S. Levitte et al. UMPS-1 and gut granule size
FEBS Journal 277 (2010) 1420–1439 ª 2010 The Authors Journal compilation ª 2010 FEBS 1439