The PA-TM-RING protein RING finger protein 13 is
an endosomal integral membrane E3 ubiquitin ligase
whose RING finger domain is released to the cytoplasm
by proteolysis
Jeffrey P. Bocock
1
, Stephanie Carmicle
1
, Saba Chhotani
1
, Michael R. Ruffolo
1
, Haitao Chu
2
and
Ann H. Erickson
1
1 Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC, USA
2 Department of Biostatistics, University of North Carolina, Chapel Hill, NC, USA
Proteins of the PA-TM-RING family have a protease-
associated (PA) domain and a RING finger domain
separated by a transmembrane (TM) domain. PA
domains are 120–210 amino acid sequences located in
the noncatalytic regions of diverse proteases [1,2]. They
are found in multiple members of MEROPS peptidase
Keywords
E3 ubiquitin ligase; neurite outgrowth;
protease-associated domain; proteolysis;
RNF13
Correspondence
A. Erickson, Department of Biochemistry

RNF13 from a membrane anchor thus provides unique spatial and tempo-
ral regulation that has not been previously described for an endosomal E3
ubiquitin ligase.
Abbreviations
APP, Alzheimer’s precursor protein; AtRMR1, Arabidopsis thaliana receptor homology region-transmembrane domain-RING-H2 motif protein;
CHO, Chinese hamster ovary; C-RZF, chicken RING zinc finger; CTF, cytoplasmic C-terminal fragment; EEA1, early endosomal antigen 1; ER,
endoplasmic reticulum; GRAIL, gene related to anergy in leukocytes; HA, hemagglutinin; HAF, hemagglutinin and 3· FLAG epitopes; HRP,
horseradish peroxidase; ICD, intracellular domain; LAMP2, lysosomal-associated membrane protein 2; MPR, mannose 6-phosphate receptor;
MVB, multivesicular body; NLS, nuclear localization signal; PA, protease-associated; PDI, protein disulfide isomerase; PNGase F, peptide:
N-glycosidase F; RNF13, RING finger protein 13; TM, transmembrane.
1860 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
families [3], including the transferrin receptor, a cata-
lytically inactive protease, prostate-specific membrane
antigen [4], the human Golgi ⁄ endosomal signal pepti-
dase peptidase-like proteins SPPL2a and SPPL2b [5],
and streptococcal C5a peptidase [6]. PA domains have
been proposed to serve as substrate or ligand recogni-
tion domains [1] or as protease regulatory regions [2],
yet they have been functionally characterized only in
plant proteins. The BP-80 receptor, which targets pro-
teases to the plant lytic vacuole through recognition of
the NPIR sorting determinant, contains a PA domain.
Binding of vacuolar proteases requires the PA domain
as well as other regions of the BP-80 luminal domain
[7].
RING finger proteins constitute a subfamily of the
proteins that possess a pattern of cysteine and histidine
residues that chelate zinc ions. The RING subfamily is
thought to function exclusively in protein–protein
interactions rather than protein–nucleic acid interac-

firmed to be necessary for induction of T-cell anergy
[16,17]. RING finger protein 13 (RNF13) was first
designated chicken RING zinc finger (C-RZF), a pro-
tein upregulated when chicken embryo brain cells were
treated with the extracellular matrix component tenas-
cin-C [18]. The protein was also upregulated in basilar
papilla when chickens were exposed to acoustic trauma
[19]. A truncated splice variant that lacks a complete
RING-H2 domain was additionally identified in mice
[19] but was not characterized. On the basis of immu-
nofluorescence microscopy and nuclear fractionation
experiments, Tranque et al. [18] reported that RNF13
is a nuclear protein, even though the tmpred algo-
rithm [20] predicts that it has a TM domain. A recent
study established that RNF13 is an E3 ubiquitin ligase
whose expression is increased in pancreatic ductal ade-
nocarcinoma tissues, suggesting that the protein may
participate in pancreatic cancer development [21].
We show that RNF13 is synthesized as an endoso-
mal integral membrane protein rather than a soluble
nuclear protein, consistent with other members of the
PA-TM-RING family. We demonstrate that RNF13
mRNA is upregulated following initiation of neurite
outgrowth, thus expanding on an array study that
found RNF13 expression to be sufficient to induce
neurite outgrowth [22]. We show that RNF13 is sub-
ject to unexpected proteolysis that releases both the
PA domain and the RING domain from the mem-
brane, providing a biochemical basis for understanding
the regulation of this family of multimodular endo-

by the algorithm pestfind [27] to be a PEST domain.
PEST domains, defined as hydrophilic stretches of at
least 12 amino acids having a high concentration of
proline, glutamic acid, serine, and threonine, are pro-
tein domains that direct rapid degradation and thus
are usually found in proteins with a short half-life [28].
The remainder of the C-terminal region is rich in ser-
ine residues, similar to transcription factor activation
domains. Multiple phosphorylation sites are predicted
in the cytoplasmic half of the protein both by netph-
osk1.0 [29] and by group-based phosphorylation
scoring (GPS) 1.1 [30,31].
Sequence alignment of RNF13 with other
PA-TM-RING proteins
Three PA-TM-RING proteins, plant AtRMR1, mouse
GRAIL and mouse RNF13, exhibit little overall
sequence identity, as shown in the alignment in Fig. S1.
Only approximately 12% of the amino acids are
identical between the three proteins, as determined by
tcoffee alignment [32]. Most of the conserved residues
(gray boxes) lie within either the PA domain or the
RING-H2 domain.
RNF13 is an E3 ubiquitin ligase
RING finger sequences frequently mediate ubiquitin
ligase activity [9]; however, at least three distinct roles
have been described for RING domains [33]. We there-
fore investigated whether the RING domain in the
cytoplasmic half of RNF13 was capable of catalyzing
polyubiquitination. The cytosolic domain of
RNF13D1–205 comprising residues 206–381, and thus

fied commercial E1 enzyme, and a commercial E2
enzyme, either UbcH5a, UbcH5c, or UbcH6, it was
able to catalyze the formation of polyubiquitin chains,
as shown by the appearance of a high molecular mass
ladder of protein bands (Fig. S2C, lanes 1–3). As there
were only four proteins present in this in vitro assay,
and one of them was ubiquitin itself, these data sug-
gest that, like many E3 ubiquitin ligases, RNF13 can
ubiquitinate itself. All three E2s assayed interacted
with RNF13, but UbcH6 appeared to produce more
polyubiquitination (Fig. S2C, lane 3). As expected by
analogy with c-Cbl, purified RNF13D1–205 C266A
was unable to catalyze polyubiquitination when added
to a similar assay (Fig. S2C, lanes 4–6), as seen by the
PA
NLS
RING Ser-RichPESTTM
HA
3XFLAG
56
162
182
240
284
307
Fig. 1. RNF13 is a PA-TM-RING protein composed of several domains that might regulate other proteins. RNF13 is predicted by TMPRED [20]
to be a TM protein, with the hydrophobic TM domain falling in the middle of the amino acid sequence (residues 182–203). Additional major
domains include a predicted signal peptide (residues 1–34), a luminal PA domain (residues 56–162), and a cytoplasmic RING-H2 domain (resi-
dues 240–292). The protein is also predicted to have an NLS (residues 214–221 or 227–233), a PEST sequence (residues 284–307), and a
serine-rich region predicted to be phosphorylated (residues 309–381). We prepared expression constructs containing one or more of the

GHI
DF
C
E
Fig. 2. Endogenous, transiently expressed and stably expressed RNF13 all show punctate staining consistent with localization to endoso-
mal–lysosomal vesicles. (A, B) Primary cortical neurons prepared from embryonic day 14.5 mouse embryos were treated with MG132 for
12 h. Endogenous RNF13 was detected with antibodies directed against the 14 amino acid C-terminal peptide of mouse RNF13. Staining
was observed with the use of secondary donkey anti-rabbit Alexa Fluor 488 serum. The size bar in (B) represents 10 lm. (C) PC12 cells sta-
bly expressing RNF13 were treated with MG132 for 12 h. RNF13 expression was detected with mouse anti-FLAG serum and, as secondary
antibody, donkey anti-mouse Alexa Fluor 568 serum. (D–F) COS cells were transiently transfected with the RNF13 expression plasmid
pSG5X-RNF13 FLAG377. RNF13 (D) was detected with rabbit anti-FLAG serum and, as secondary antibody, anti-rabbit Texas Red serum.
Cells were counterstained with mouse antibodies raised against PDI (E) and donkey anti-rabbit Alexa Fluor 488 serum. These panels are
merged in (F). The size bar in (D–F) represents 20 lm. (G–I) HeLa cells stably expressing RNF13 were treated with MG132 for 12 h. RNF13
(G) was stained with mouse anti-FLAG and donkey anti-mouse Alexa Fluor 488 sera. Calnexin staining (H) was observed with rabbit anti-
calnexin and goat anti-rabbit Alexa Fluor 568 sera. These panels were merged in (I). The size bar in (G–I) represents 5 lm. RNF13 did not
colocalize with either of the two ER markers.
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1863
RNF13 observed in embryonic mouse cortical neu-
rons using an antiserum specific for the C-terminal 14
amino acids of RNF13 showed punctate, non-nuclear
staining characteristic of endosomes and lysosomes
(Fig. 2A,B). To facilitate detection of RNF13 by
immunofluorescence and to enable us to determine the
origin of the biosynthetic forms detected by western
blotting, we constructed vectors to express RNF13
with an N-terminal hemagglutinin (HA) epitope and a
C-terminal FLAG tag. Stably expressed, epitope-
tagged RNF13 exhibited punctate staining in PC12
cells, which are derived from a pheochyromocytoma of

Marker
Fig. 3. RNF13 is localized in MVBs and
endosomes. COS cells (A–L) or HeLa cells
(M–O) were transiently transfected with
RNF13-FLAG377, which was detected using
rabbit anti-FLAG sera (B, E, H, K, N). Cells
were costained with mouse anti-human
Golgin 97 (A) serum, mouse anti-human
LAMP2 serum (D), mouse anti-human CD63
serum (G), mouse anti-human MPR serum
(J) or mouse anti-human EEA1 serum (M).
Primary antibodies were visualized with the
secondary antibodies donkey anti-mouse
AlexaFluor 488 serum (A, D, G, J), goat
anti-rabbit Texas Red serum (B, E, H, K),
donkey anti-rabbit AlexaFluor 488 serum (N)
and goat anti-mouse AlexaFluor 568 serum
(M). RNF13 colocalized with LAMP2 (F),
CD63 (I), and MPR, (L), but not with Gol-
gin 97 (C) or EEA1 (O). Images were
obtained with a Zeiss LSM 210 confocal
microscope. The size bars represent 10 lm
(A–C, J–L) and 20 lm (D–I).
Proteolytic regulation of RNF13 J. P. Bocock et al.
1864 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
two different ER chaperone proteins. RNF13 did not
colocalize with endogenous protein disulfide isomerase
(PDI) when expressed transiently in COS cells
(Fig. 2D–F). Similarly, RNF13 expressed stably in
HeLa cells did not colocalize with calnexin (Fig. 2G–

To characterize the biosynthetic processing of RNF13,
we constructed viral expression vectors encoding
mouse RNF13 with an HA epitope at position 38 and
a3· FLAG epitope at position 381 (RNF13-HAF)
that we used to infect Chinese hamster ovary (CHO)
cells to produce the CHO-RNF13-HAF cell line, which
stably expresses RNF13. FLAG-positive RNF13-spe-
cific bands were not detected by western blot analysis
of cells expressing empty vector (Fig. 4A, lane 1). Sur-
prisingly, RNF13-specific FLAG-positive bands were
barely detectable in cell lysate when cells stably
expressing RNF13 were treated with dimethylsulfoxide
vehicle for 8 h (Fig. 4A, lane 2). When these cells were
incubated with the protease inhibitor MG132 in
dimethylsulfoxide for 8 h prior to harvest, however, a
specific RNF13 banding pattern indicative of extensive
post-translational modification was reproducibly
45” NS
1 2
1 2 3
-
-
-
1
2
3
97
37
54
kDa

35
S]methionine for 45 min. RNF13 was immunoprecipitated with anti-FLAG serum and
resolved on a 12% polyacrylamide gel (lane 1). To detect nonspecific protein bands, normal whole serum (NS) was substituted for specific
affinity-purified anti-FLAG serum (lane 2).
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1865
detected (Fig. 4A, lane 3). The pattern included a het-
erogeneous collection of proteins of approximately
80 kDa, a second series of proteins that occasionally
resolved into four discrete bands at approximately
65 kDa (e.g. Fig. 5A, lane 2), three protein bands of
approximately 45 kDa, and one protein band of
approximately 36 kDa. As all these proteins were visu-
alized with antiserum that recognizes the 3· FLAG
epitope at residue 381, they all contain the C-terminus
of RNF13. An identical protein pattern was obtained
when RNF13-HAF was expressed stably in B35 rat
neurons (data not shown).
We next utilized antiserum specific for the N-termi-
nal HA epitope tag (residue 38) to determine which of
the RNF13 proteins in the banding pattern contain the
N-terminus. Cells were treated as indicated (Fig. 4B).
The specific proteasome inhibitor epoxomicin stabi-
lized RNF13 (Fig. 4B, lane 5), as did MG132 (Fig. 4B,
lane 4). Both the heterogeneous bands at  80 kDa
and the group of bands at  65 kDa were recognized
by the anti-HA serum (Fig. 4B, lanes 4 and 5). As
these proteins are also recognized by the anti-FLAG
serum, they must possess both residues 38 and 381 and
therefore be close to full-length RNF13. The lower

dase treatment, the two upper protein bands
disappeared, with a concomitant increase of the lowest
band. Identical results were obtained with drug prepa-
rations from two different suppliers (Fig. 5A, lanes 3
and 4). To confirm this result, CHO cells transiently
expressing FLAG-tagged RNF13 were cultured in the
presence of tunicamycin, an antibiotic that inhibits
transfer of N-acetylglucosamine 1-phosphate to doli-
cholmonophosphate [37], thus blocking the synthesis
of asparagine-linked oligosaccharide chains on glyco-
proteins. Tunicamycin treatment reproducibly reduced
the amount of the upper band and resulted in loss of
the middle band. These results confirm that two
N-linked sugar chains can be removed from RNF13,
supporting the predictions made by netnglyc 1.0.
As a percentage of certain integral membrane pro-
teins, such as the Alzheimer’s precursor protein (APP)
[38] and the immunoglobulin invariant chain [39,40],
acquire chondroitin sulfate glycosaminoglycan chains,
we also assayed RNF13 for this modification. RNF13
possesses one potential Ser-Gly dipeptide acceptor
sequence [41] in its luminal domain. When immuno-
precipitated RNF13 was treated with chondroitin-
ase ABC, the intensity of the diffusely staining bands
1 2 3 4 A
B
5
–+
Chondroitinase
Transfection +

the absence (lane 1) or presence (lane 2) of chondroitinase ABC
prior to resolution on a 12% polyacrylamide gel.
Proteolytic regulation of RNF13 J. P. Bocock et al.
1866 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
at 80 kDa dramatically decreased, whereas the 65–
70 kDa bands increased in intensity (Fig. 5B). This
result indicates that at least a proportion of the
RNF13 protein is modified with chondroitin sulfate.
Proteolysis releases N-terminal and C-terminal
fragments of RNF13 from the membrane
To further characterize the 36 kDa FLAG-positive
RNF13 band observed in cell lysates, CHO cells were
transfected transiently with a construct that encodes
only the C-terminal half of RNF13. This variant
(RNF13D1–204) is initiated a few residues beyond the
putative TM sequence and retains the FLAG epitope.
It was found to comigrate with the 36 kDa protein in
cell lysates (Fig. 6A, lane 2 versus lane 4), suggesting
that the 36 kDa band is derived from full-length
RNF13 by proteolysis at or near the TM sequence. An
HA-positive protein of approximately the same size
was reproducibly detected when blots probed with
anti-HA serum were overexposed (Fig. 6B, lane 5).
This protein band always appeared fuzzy, consistent
with the presence of carbohydrate. Detection of this
protein suggests that the N-terminal domain of
RNF13, like the C-terminal domain, is released by
proteolysis from the TM anchor localized approxi-
mately in the middle of the protein.
RNF13 is a type I integral membrane protein

To confirm that RNF13 is an integral, not a periph-
eral membrane protein, we isolated microsomes from
CHO-RNF13-HAF cells and lysed them in high-pH
carbonate buffer (Fig. 7B). Freezing and thawing
microsomes in pH 11.5 buffer lyses vesicles and solu-
bilizes peripheral membrane proteins not embedded in
the membrane bilayer [42,43]. The luminal lysosomal
protease cathepsin L, detected as a control, was
present in the soluble fraction, confirming that soluble
content proteins are released by carbonate treatment
(data not shown).
All forms of RNF13 present in microsomes and
recognized by the anti-FLAG serum were present in the
membrane fraction and were not solubilized when vesi-
cles were lysed at high pH, indicating they are integral,
not peripheral, membrane proteins (Fig. 7B, lane 2).
The HA-tagged 36 kDa fragment of RNF13 was also
detectable within microsomes (Fig. 7B, lane 3), sug-
gesting that the luminal domain is shed within endo-
somes. With this protocol, an additional HA-positive
protein in the RNF13 pattern was reproducibly detect-
Ctl ICD RNF13
RNF13
12 3 4
5
Anti-FLA
ABnti-HA
-
-
GA

absence of the epitope tags, is presented in Fig. 7C.
The three cytoplasmic C-terminal fragments (CTFs) or
‘stubs’ remaining after loss of the PA domain could be
generated by multiple proteases or could result from
multiple cleavages by one enzyme. Other biosynthetic
intermediates may be present but not detectable by our
gel system. Additionally, the ratio of the forms may
vary with the cell type and the metabolic condition of
the cells expressing RNF13.
Inhibiting the vacuolar ATPase only partially
stabilizes RNF13
Since RNF13 localizes to the endosomal–lysosomal
membrane system, we treated cells with two inhibitors
that raise the pH of vesicles in an attempt to inhibit
lysosomal proteolysis of RNF13. Bafilomycin A1
inhibits the vacuolar ATPase [44,45], and ammonium
chloride raises the pH of lysosomes and blocks the
light–heavy chain cleavage of lysosomal cathepsin L
[46]. Although MG132 is commonly employed as a
proteasome inhibitor, it has also been reported to
inhibit lysosomal cathepsins [47,48], calpains [47],
and BACE1 [49]. Stably expressed RNF13 was barely
detectable in cell extracts unless the cells were pretreated
with MG132 (Fig. 8A, lane 1 versus lane 2). Inhibition
of the vacuolar ATPase with bafilomycin A1 or by
treating cells with ammonium chloride (Fig. 8A, lanes
3 and 4, respectively) stabilized biosynthetic forms of
RNF13, but not as efficiently as did MG132 treatment
of cells. The data suggest that other proteases primar-
ily mediate the turnover of RNF13 in vesicles distinct

G
FLAG
FLA
G
70
FLAG
FLA
G
Full-length CTF ICD
~kDa:
63
97-
54-
37-
-
-
-
97
54
37
36
3
-
-
-
-
95
72
55
36

including throughout tissues of the human immune
and nervous systems [50]. However, initial northern
blot analysis of expression of C-RZF, the chicken
homolog of RNF13, showed that the protein was
expressed in embryonic heart and brain, but not in
liver [18]. We therefore analyzed mouse RNF13
expression by quantitative real-time RT-PCR, isolating
mRNA from both embryonic and adult mouse tissues.
The oligonucleotides used in this assay were specifi-
cally designed to bind only the full-length RNF13
transcript. Expression of RNF13 in adult heart tissue
was similar to that in spleen. We observed fold
increases of 5.7, 2.6 and 1.9 for adult kidney, liver and
brain, respectively, relative to spleen (Table 1; see
Table S3 for statistical analysis). The PA-TM-RING
family member GRAIL has been found to have similar
expression in mouse tissues, using northern blots [15],
but it has been primarily studied in T-cells. We also
observed that RNF13 expression levels in adult tissues
were higher than in the corresponding embryonic
tissue. For example, there was a four-fold increase in
adult brain as compared to embryonic brain after 14.5
or 16.5 days of development (Table 1). Our analysis of
embryonic tissue showed similar expression of RNF13
1 2 3 4
+MG132
+Bafilomycin
+DMSO
+NH
4

in both liver and brain, consistent with our findings in
adult tissue.
RNF13 is upregulated during neurite outgrowth
Quantitative real-time RT-PCR data indicated that
RNF13 is expressed in brain tissue (Table 1). A previ-
ous array study identified the RNF13 gene as one of
five genes that were able to promote neurite extension
when expressed ectopically in PC12 neuronal precursor
cells cultured on collagen [22]. The level of RNF13
expression was not assayed under these conditions. We
therefore determined whether endogenous RNF13 gene
expression increases during neurite outgrowth. B35
neuroblastoma cells were treated with dibutyryl-cAMP,
a reagent that stimulates neurite extension by these
cells [51], and RNF13 expression was analyzed using
quantitative real-time RT-PCR (Fig. 10). We observed
a two-fold increase in RNF13 mRNA after 72 h of
outgrowth. A similar two-fold increase of RNF13
expression was observed after 5 days of outgrowth.
Thus, RNF13 ubiquitin ligase activity may play a role
in the regulation of nerve cell development.
Discussion
We have determined that endosomal membranes pos-
sess an E3 ubiquitin ligase that can be released into
the cytoplasm by proteolysis. The PA-TM-RING pro-
tein RNF13 is synthesized as an integral endosomal
membrane protein, but post-translational proteolysis
solubilizes the C-terminal half of this type I membrane
protein. The role of ubiquitin addition in receptor
endocytosis and in the formation of multivesicular

ized using 18S rRNA as an internal control. Each RNA sample was
analyzed in duplicate, using quantitative real-time RT-PCR. Stati-
stical analysis of the data is presented in Table S3.
Group Organ
Mean
DCT
Fold
expression
Adult Brain 9.4 2.9
Adult Heart 10.3 1.5
Adult Kidney 8.1 6.7
Adult Liver 9.1 3.6
Adult Spleen 10.9 1.0
Adult Liver 9.1 11.5
E14.5 Liver 12.6 1.0
E16.5 Liver 11.2 2.7
Adult Brain 9.4 5.5
E14.5 Brain 11.8 1.0
E16.5 Brain 11.3 1.5
Fig. 10. RNF13 expression is increased following induction of neu-
rite outgrowth. B35 cells were plated on dishes coated with fibro-
nectin (5 mgÆmL
)1
) and incubated with dibutyryl-cAMP (100 lM)to
induce neurite outgrowth. RNA was harvested using Trizol reagent,
each plate being harvested separately as a single sample. Sample
numbers of n = 9 were collected for control cells, and sample num-
bers of n = 6 were collected for both time points of outgrowth.
Quantitative real-time RT-PCR was performed in duplicate for each
RNA sample, using ABI PRISM 7700 hardware and software. Data

atic RNF13 [21]. Interestingly, a splice variant of
human RNF13 (NM183383) has been identified that is
equivalent to this portion of RNF13.
Ectodomain shedding must precede proteolytic
cleavage within the membrane. The soluble HA-tagged
RNF13 fragment detected within microsomes, which is
approximately the size of the PA domain, is probably
primarily degraded in lysosomes, but it could be
secreted if the endosomes fuse with the plasma mem-
brane. A gene product equivalent to the PA domain
has been identified in mammalian cells (hPAP21), but
its physiological function is unknown [59]. The locali-
zation of RNF13 to MVBs or late endosomes sug-
gested that its N-terminal proteolysis might be
mediated by lysosomal enzymes. However, isoforms of
RNF13 were only slightly stabilized by the addition of
reagents that inhibit lysosomal proteases. Thus, other
cellular proteases must be primarily responsible for
mediating the turnover of RNF13.
RING E3s are commonly cytosolic, nuclear or
peripheral membrane proteins. The initial study of
RNF13 [18] concluded that the protein is localized in
the nucleus, on the basis of both immunofluorescent
staining and cell fractionation. The validity of the
fractionation protocol utilized has been questioned [1],
however, and the tmpred algorithm predicts that
RNF13 has a TM sequence. Consistent with this, our
analysis of cellular membranes washed with high-salt
solution establishes that RNF13 is indeed an integral
membrane protein. This is consistent with data showing

half-life that is tightly regulated by ubiquitination and
proteasome degradation; (b) RNF13 may need a specific
signal to move into the nucleus either on its own or with
the help of an adaptor protein that facilitates entry into
or retention in the nucleus – it is possible that the adap-
tor protein is more abundant in certain cell types, or is
upregulated in response to an external stimulus, and
thus RNF13 cannot effectively move into the nucleus in
cultured cells; and (c) alternatively, RNF13 may not go
to the nucleus and instead may function as an E3 ligase
in the cytoplasm. Precedents exist for all three possibili-
ties. The ICDs of proteins such as Notch and APP are
notoriously difficult to detect in nuclei, as they represent
a small percentage of the whole protein and they are
very labile, so it remains possible that we are simply
unable to detect the small amount of ICD that might
localize to the nucleus constitutively. The APP ICD
must form a complex with the nuclear adaptor Fe65 and
the histone acetyltransferase Tip60 in order to target to
the nucleus [61,62]. Other ICDs function exclusively in
the cytoplasm. The adhesion protein N-cadherin under-
goes processing by c-secretase to generate an epsilon
cleavage product, N-Cad ⁄ CTF2, which forms a complex
J. P. Bocock et al. Proteolytic regulation of RNF13
FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS 1871
in the cytoplasm with the transcription factor CBP, pro-
moting CBP ubiquitination, proteasomal degradation,
and repression of CBP ⁄ CREB transcription [63].
The PA-TM-RING proteins GRAIL [15], Goliath-
related E3 ubiquitin ligase 1 [64], h-Goliath [57] and

and neurite outgrowth. Although RNF13 homologs
have been identified in humans, dog, chicken, fruit fly,
mosquito, tobacco and rice, no PA-TM-RING protein
has been identified in yeast, suggesting that this E3
activity may be necessary to modulate a function
unique to multicellular organisms, such as cell–cell
interaction or tissue development.
Experimental procedures
Reagents
Bovine fibronectin, dibutyryl-cAMP, chondroitinase ABC,
MG132 and bafilomycin A1 were obtained from Sigma-
Aldrich (St Louis, MO, USA). Lipofectamine 2000 was
from Invitrogen (Carlsbad, CA, USA) and FuGENE 6
transfection reagent was from Roche Diagnostics (Indiana-
polis, IN, USA). Bicinchoninic acid reagents were from
Pierce/Thermo Fisher Scientific (Rockford, IL, USA). Flu-
orsave was from Calbiochem/EMD Chemicals (Gibbstown,
NJ, USA). Purified ubiquitin was a gift from J. McCarville
(UNC-CH). Rabbit E1 enzyme and human UbcH5a,
UbcH5c and UbcH6 E2 enzymes were from Boston
Biochemicals (Cambridge, MA, USA).
Antibodies
Affinity-purified polyclonal rabbit anti-RNF13 serum was
prepared by GenScript Corp (Piscataway, NJ, USA). using
the peptide CPNGEQDYNIANTV, the 14 C-terminal resi-
dues of mouse RNF13. Mouse and rabbit anti-FLAG IgG
sera, horseradish peroxidase (HRP)-conjugated anti-FLAG
M2 IgG1 serum and HRP-conjugated anti-HA IgG1 serum
were from Sigma-Aldrich. Sheep anti-mouse HRP-conju-
gated serum was from Amersham/GE Healthcare (Chalfont

a C-terminal 3· FLAG tag added by cloning into p3·
FLAG vector (Sigma-Aldrich). The expression and target-
ing of the protein with the 3· FLAG epitope appeared to
be indistinguishable from those of the protein modified
with a 1· FLAG epitope inserted after residue 377
(Fig. 2). The oligonucleotides described in Table S1 were
Proteolytic regulation of RNF13 J. P. Bocock et al.
1872 FEBS Journal 276 (2009) 1860–1877 ª 2009 The Authors Journal compilation ª 2009 FEBS
purchased from Integrated DNA Technologies (Coralville,
IA, USA), Invitrogen, and the Nucleic Acids Core
Facility, UNC-CH. Automated sequencing was performed
at the UNC-CH Genome Analysis Facility.
Bacterial expression and purification
BL21DE3 bacteria were transformed with pET3E-His
vectors encoding mouse RNF13D1–205 or RNF13D1–205
with a C266A point mutation. Concentrations of purified
protein were determined using a bicinchoninic acid assay
according to the manufacturer’s instructions. Purified
protein was resolved on both 12% and 15% polyacrylamide
gels to assess purity; 15% gels were analyzed by Coomassie
stain, and 12% gels were western blotted with anti-6· His
serum. Relative amounts of protein were determined by
using densitometry software (kodak 1d, version 3.6.2,
Eastman Kodak, Rochester, NY, USA).
In vitro ubiquitin ligase assay
To perform the ubiquitin ligase assay, we used a reaction
mixture consisting of 50 mm Tris ⁄ HCl (pH 7.6), 2 mm
MgCl
2
,2mm ATP, 1 mm dithiothreitol, 100 nm rabbit E1

4
Cl (10 mm)
was added for 24 h, and bafilomycin A1 (1 lm) for 8 h.
When specified, RNF13 was immunoprecipitated from cells
and resolved on polyacrylamide gels as described previously
[46].
High-mannose sugar addition was inhibited by treatment
of cells with tunicamycin (Roche) at 1.5 lgÆ mL
)1
for 14 h.
N-linked sugar was removed by treating immunoprecipi-
tates with PNGase F (Roche and NE BioLabs, Ipswich,
MA, USA) according to the manufacturers’ instructions.
Chondroitinase
Immunoprecipitated protein eluted from resin in
SDS ⁄ PAGE loading buffer was divided into two equal
parts. After addition of pH 7 sodium acetate to a final con-
centration of 50 mm, chondroitinase ABC (0.2 U) or an
equivalent volume of SDS ⁄ PAGE buffer was added as
specified. Samples were incubated at 37 °C overnight. Dith-
iothreitol (50 mm) and bromophenol blue were added to
each tube, and proteins were resolved by 12% SDS ⁄ PAGE
and visualized by western blotting as indicated.
Cell fractionation
Cell fractionation was performed as described previously
[46], with addition of 10 mm sodium fluoride, 10 mm
sodium vanadate and 10 mm sodium pyrophosophate to
the homogenization buffer. Soluble cytoplasmic proteins
and microsomes were separated by centrifugation for 1 h at
60 000 g and 4 °C in a Beckman TL-100 centrifuge. The

ments 2.0 and adobe illustrator.
Quantitative real-time RT-PCR analysis of RNF13
mRNA expression
Total RNA was harvested from adult and embryonic
mouse tissues and from cultured cells using TRIzol reagent
(Invitrogen) according to the manufacturer’s instructions.
Embryonic mice tissues were harvested at embryonic days
14.5 and 16.5. B35 neuroblastoma cells were cultured on
dishes coated with 5 lgÆmL
)1
fibronectin, and induced to
extend neurites by incubation with 100 lm dibutyryl-cAMP
for 3 or 5 days [51]. Quantitative real-time RT-PCR was
performed using the ABI PRISM 7700 Sequence Detection
System. RNF13 mRNA was normalized using 18S rRNA
as an internal control. The sequences of the oligonucleo-
tides and probes used are given in Table S2. Probes were
synthesized with a 5¢-FAM dye. These oligonucleotides and
probes were designed and synthesized by the UNC-CH
Animal Clinical Chemistry and Gene Expression Laborato-
ries. RNF13 oligonucleotides were used at 0.1 lgÆlL
)1
; 18S
oligonucleotides were used at 20 ngÆlL
)1
. RNF13 and 18S
probes were used at 20 lm. One hundred nanograms of
RNA was used for all reactions. The cycling parameters
used were as follows: 48 °C for 30 min, 95 °C for 10 min,
and 40 cycles of 95 °C for 15 s, followed by 60 °C for

supported by NIH SPIRE Postdoctoral Fellowship
GM000678.
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Supporting information
The following supplementary material is available:
Fig. S1. tcoffee alignment [32] of murine RNF13
(mRNF13) (NCBI Locus AAH58182) with PA-TM-
RING proteins murine GRAIL (mGRAIL) (NCBI
Locus NP_075759) and A. thaliana (AtRMR1) (NCBI
Locus AAF32326).
Fig. S2. RNF13 has ubiquitin ligase activity in vitro.
Table S1. Oligonucleotides used for cloning RNF13
into vectors, introducing mutations and deletions, and
adding epitope tags.
Table S2. Oligonucleotides used for quantitative real-
time RT-PCR.
Table S3. Statistical analysis of RNF13 expression
data quantitated by real-time RT-PCR.