Letter reSeArCH
563 to 910, hereafter denoted SdeA(563–910))^11 that was purified
from HEK293T cells expressing GFP–SidJ (Flag–mART) also failed
to ubiquitinate Rab33b with the PDE-competent SdeA(E860A/E862A)
mutant^3 ,^4 (Fig. 2c). We therefore conclude that SidJ targets the mART
activity of SdeA.
Analysis by liquid chromatography coupled with tandem
mass spectrometry (LC–MS/MS) identified a mass shift of 129.04 Da
(m/z = 129.04, z = 1) on the peptide –H 855 GEGTESEFSVYLP
EDVALVPVK 877 – in Flag–mART (Fig. 2d, e). The modification—
probably the addition of a glutamate—was mapped to E860, one of the
catalytic residues of the mART^2 (Fig. 2e). Approximately 93.7% of E860
was modified in samples coexpressed with GFP–SidJ, and a modifica-
tion of 258.09 Da (m/z = 258.09, z = 1)—presumably diglutamate—
was also detected on a small portion of the same peptide (Extended
Data Fig. 1). Thus, SidJ may be a glutamylase that ligates one or more
glutamate moieties to E860 of SdeA.
We did not detect SidJ activity in reactions containing ATP—the
energy source for known glutamylases^12 —and l -glutamate, or its struc-
tural isomers N-acetylserine and N-methylaspartate (Extended Data
Fig. 2a). Because the inhibitory effects of SidJ are evident only when it is
expressed in mammalian cells, we tested the hypothesis that its activity
requires one or more factors of eukaryotic origin by including lysates
of E. coli or HEK293T cells in the reactions. Lysates of HEK293T cells
(native or boiled) caused a decrease in Rab33b modification (Extended
Data Fig. 2b), which indicates that one or more heat-stable factors
specific to eukaryotic cells are required for the activity of SidJ.
Analysis of the Pfam database^13 of protein families revealed an
IQ-like motif, which is involved in CaM binding, located near the
carboxyl end of SidJ (Fig. 3a). The yeast toxicity of SidJ^14 was sup-
pressed by mutations in I841 and Q842—two residues in IQ motifs
that are important for CaM binding^15 —or by overexpression of the
yeast CaM gene cmd1 (Fig. 3b), thereby validating the nature of the IQ
motif. Indeed, binding between SidJ and CaM occurred in cells that
were infected with relevant L. pneumophila strains or that coexpressed
these two proteins, and the IQ motif was found to be required for
optimal binding (Fig. 3c, d).
CaM, and SidJ together with l -glutamate—but not the two gluta-
mate isomers—abolished SdeA-mediated ubiquitination (Fig. 3e).
Consistent with the heat-insensitivity seen in mammalian cell lysates,
boiled CaM was partially active (Extended Data Fig. 2c). Notably,
we found that SdeA can be modified by^14 C-glutamate only in reac-
tions containing CaM, and that SdeA(E860A) cannot be modified
by^14 C-glutamate, establishing that E860 is the major modification
site (Fig. 3f). Similar to other glutamylases^12 , ATP binds SidJ (with a
dissociation constant, Kd, of 1.45 μM) and is required for SidJ activity
(Extended Data Fig. 2d, e). CaM-dependent inhibition by SidJ was
found for all members of the SidE family (Extended Data Fig. 2f).
Under our experimental conditions, 0.006 μM and 0.055 μM of CaM
was required to activate SidJ and SidJ(I841D/Q842A) respectively; this
explained the observation that SidJ(I841D/Q842A) still complemented
the phenotype associated with the ∆sidJ mutant (Extended Data
Fig. 3). Together, these results establish that SidJ is a CaM-dependentWild typeVectorI841D/Q842AWild typeVectorI841D/Q842AGlucoseGalactose
SidJ + vector
sidJ + cmd1sidJ + vectorGlucoseGalactoseIB: SidJIB: PGK1VectorWild typeI841D/Q842AIB: SidJ
IB: PGK1sidJ+ vector$,7,45):5($5.1
,,.,45):5(+..(
$7.,4$6)5*+,75
$7/,4.,<5*:.&5
$9,,4.595*:/$5
0)4,4565.)'(35
,,.,4$),5$1.$5
,..946)/5*:/&5
$9,,45$<55+//.
$,,,45$<55<//.
11(94/()5.6:(5838
773
35
708
866
495
839
206
1919
1909
406ab100
50150eL-glutamate
N-acetylserine
N-methylaspartate
CaM++IB: FlagIB: SdeAATP, His6-SdeA, GST–SidJ
(4×Flag–Rab33b, Ub, NAD+)Ub-4×Flag–Rab33b4 ×Flag–Rab33b37fGST–SidJ
GST–SidJ(D542A/D545A)
His 6 -SdeA
His 6 -SdeA(E860A)
CaM(^14) C-Glutamate
- – – – – +
- – + – – +
- – + – + +
- – – + + +
- – – – +
- – – +
- – + +
- – + + +
- – – – +
150
100
75
50
37
25
20
15SdeA or
SdeA(E860A)
SidJ or
SidJ(D542A/D545A)CaM(^14) C-Glu-SdeA
150
250
100
75
50
37
25
20
15
IB: SidJ 150
sidJ + cmd1
sidJ
- cmd1
kDa
100
50
kDa
d
IB: tubulin
IB: SidJ
Translocation
Pull-down:
IB: SidJ CaM
Wild typedotA
- ΔsidJΔsidJ
(pSidJ)100100
50ExpressionIB: SidJ(^50) IB: ICDH
100
kDa
kDa
kDa
SidJ (L. pneumophila) YP096168.1
SdjA (L. pneumophila) YP096515.1
Neuromodulin (human) P17677
Myosin Ib (mouse) AAH54786. 1
Myosin Va (human) NP_000250
Protein kinase C epsilon (human) Q02156
Ras GAP-related protein (human) P46940
CDC25 (human) AAA58417
Sodium channel protein I (human) P35498
Sodium channel protein II (human) S29185
GLP1R (human) AAA6378 7
c
GFPGFP–SidJ
TC
L
Pull-down: CaM
IB: GFP
GFP–SidJ
GFP–SidJ
GFP
100
50
75
37
25
100
25
GFP–SidJ
kDa (I841D/Q842A)
Fig. 3 | Calmodulin is the host cofactor required for the glutamylase
activity of SidJ. a, SidJ contains an IQ motif. The list shows the alignment
of the IQ domain of SidJ with that of several CaM-binding proteins.
Conserved residues are highlighted in red. The accession numbers for
each of the proteins (from NCBI databases) are included. b, The cmd1
gene suppresses the yeast toxicity of SidJ. The top two panels show images
of serially diluted yeast cells inducibly expressing sidJ or its IQ mutant
spotted onto the indicated media for 2 days. The lower two panels show
the suppression of SidJ toxicity by cmd1. The expression of SidJ in each
strain was examined and PGK1 was detected as a loading control (right).
c, d, The interactions between SidJ and CaM. Beads coated with CaM
were incubated with lysates of macrophages infected with the indicated
bacterial strains to analyse its binding to SidJ (c, top). SidJ in bacteria
(c, middle) or translocated into the host cytosol (c, bottom) was also
examined. The bacterial isocitrate dehydrogenase (ICDH) and tubulin
were analysed as loading controls, respectively. Lysates of HEK293T
cells transfected to express GFP–SidJ or GFP–SidJ(I841D/Q842A) were
incubated with CaM-coated beads (d). SidJ or SidJ(I841D/Q842A) bound
to CaM was analysed by immunoblotting (bottom). TCL, total cell lysates.
e, Inhibition of SdeA activity by SidJ requires glutamate and CaM. CaM
was added to a subset of a series of reactions containing SdeA, GST–SidJ
and l -glutamate, N-acetylserine or N-methylaspartate. The activity of
SdeA was measured by Rab33b ubiquitination. f, SidJ is a CaM-dependent
glutamylase that modifies SdeA at E860. A series of reactions containing
the indicated proteins,^14 C-glutamate and ATP were allowed to proceed
for 2 h at 37 °C. The incorporation of^14 C-glutamate was detected by
autoradiography. Data shown in b–f are one representative of at least three
experiments with similar results.
15 AUGUSt 2019 | VOL 572 | NAtUre | 389