Nature - 15.08.2019

reSeArCH Letter

sdeC Orf2 SidJ SdeB SdeA

a)

b) c)

d)

GSTUSP2GST-SidJ

M1-Ub 2

Ub

K6-Ub 2

Ub

K11-Ub 2

Ub

K27-Ub 2

Ub

K29-Ub 2

Ub

K33-Ub 2

Ub

K48-Ub 2

Ub

K63-Ub 2

Ub

GSTUSP2GST-SidJ

UbiquitinGSTGST-SidJ

Ub-PR-Rtn4 peptide

IB:Ub

PR-Ub-Rtn4

IB:GST

GST-SidJ

GST

48-

25-

35-

63-

75-

100-

180-

Ub

SidJ

other Legionella

proteins

contaminants

Distribution of IBAQ intensities

Sid

J

Sde

A

Sde

B

Sde

C

LaiE SdeD

1.0 1004

1.0 1005

1.0 1006

1.0 1007

1.0 1008

1.0 1009

1.0 1010

1.0 1011

Abundance of PRib-Ubiquitination enzymes

IBAQ intensities

Ub-Rab33b

180

245

135

100

75

63

48

35

IB: HA

HEK293TIP: HA

HA-Ub WT

USP2GFPGFP-SidJ

GFP-SidJ

USP2

GFP

IB: His

IB: GFP

Ub-Rab33b-His

GFP VectorGFP-SidJ

135

75

63

35

25

100

48

35

25

100

75

63

48

35

25

180

135

Extended Data Fig. 1 | SidJ does not possess intrinsic deubiquitinase
activity. a, Genetic locus of sdeC-orf2-sidj-sdeB-sdeA in the Legionella
genome. b, Left, GFP–SidJ that was ectopically expressed and purified
from HEK293T cells was incubated with canonical HA–ubiquitin chains
purified from mammalian cells. The canonical deubiquitinase USP2 was
used as a positive control. Right, GFP or GFP–SidJ was incubated with
purified SdeA–ubiquitinated Rab33b. The experiment was repeated twice
independently, with similar results. c, Full-length SidJ was incubated

with various substrates modified with canonical ubiquitination or

phosphoribosyl-linked ubiquitination, to probe the cleavage activity.

USP2 was used as a positive control for cleaving canonical ubiquitin

chains. The experiment was repeated twice independently, with

similar results. d, SidJ purified from Legionella was analysed by mass

spectrometry, and protein quantification was performed using the

MaxQuant iBAQ algorithm. The experiment was repeated twice

independently, with similar results.