Nature - USA (2020-08-20)

476 | Nature | Vol 584 | 20 August 2020

Article

yeast cells by fluorescence microscopy (Fig. 1d, Extended Data Fig. 2).
The nine proteins were markedly downregulated in Emc3-knockout
cells. The downregulation is due to the absence of EMC function
rather than transcriptional variation, because the levels of mRNA of these
client genes were similar or increased compared to those
in the wild-type cells, except for the 50% reduction of Hxt3 mRNA
(Fig. 1e).
Among the 38 putative EMC clients, six (Pdr5, Pdr12, Pho90, Pma1,
Ptr2 and Snq2) were found to be associated with EMC^1 , and two (Mrh1
and Pma1) were reported to rely on EMC for membrane localization^16 ,^17.
Notably, 16 of the clients had their N termini facing outside; the others
faced the cytosol (Supplementary Table 1), which suggests that EMC
does not have a preference for the N-terminal location of the client
(inside or outside)^1 ,^9.

EMC architecture and subunit structures

We determined a 3.0-Å average resolution cryo-EM three-dimensional

(3D) map (Fig. 2a–c, Extended Data Fig. 1b–g, Extended Data Table 1,

Supplementary Video 1). The high resolution allowed us to build the

atomic model of EMC de novo (Extended Data Figs. 3, 4, Supplemen-

tary Table 3). The structure contained the previously known subunits

Emc1–Emc6 plus Emc7 and Emc10 (Figs. 1a, 2a). The EMC structure is

approximately 160 × 100 × 80 Å (Fig. 2a, b). Five subunits (Emc1 and

Emc3–Emc6) are transmembrane proteins having a total of 12 TMHs.

The remaining three subunits—Emc2, Emc7 and Emc10—are aqueous

proteins (Fig. 2c). The complex has a transmembrane region, a large

lumenal region, and a smaller cytosolic region. There are two ordered

phospholipids in the transmembrane region, one facing the lumen and

surrounded by Emc3, Emc4 and Emc6, and the other facing the cytosol

and surrounded by TMHs of Emc3, Emc5 and Emc6. We identified six

N-glycans, three in the lumenal domain of Emc1 (N73, N106 and N192)

and three in Emc7 (N53, N85 and N115) (Fig. 2b). We also observed two

disulfide bonds, one between C701 and C709 of Emc1 and the other

between C65 and C78 of Emc10. The patterns of glycosylation and

ab

kDa

135

100

80

58

46

32

25

22

17

11

Emc1

Emc2

Emc7

Emc1 0

Emc3

Emc4/5-Flag

Emc6

WT

Emc2Δ

Emc3Δ

Emc4Δ

Emc1Δ

Emc5Δ

30 °C 37 °C

Emc6Δ

Emc1 0 Δ

Emc7Δ

c

Mrh1

WT E3KO

eGFPNomarski

Ftr1

Hxt3

Fet3

WT

E3KO

eGFP

e

Relativ

e mR

NA

am

ount

(%

)

125

100

75

50

25

0

Nomarski

Pma1

Fet4

Dip5

d

Pdr5

Ena1

Fet3Mrh1Pdr5Ftr1Hxt3Ena1Dip5Fet4Pma1

3 μm

PMA1

PDR5

MRH1

HXT3

ENA1

FET3

FTR1

DIP5

FET4

0

1

2

3

4

5

Fold change

–log

( 10

P

value)

0.1250.250.5 1248

Fig. 1 | Purification of the yeast EMC and identification of EMC client proteins.
a, The Coomassie blue-stained SDS–PAGE gel of the purified EMC complex. For
gel source data, see Supplementary Fig. 1. b, Growth of tenfold serial diluted
yeast strains (wild-type (WT) and individual Emc subunit knockouts) on YPD
plates at 30 °C and 37 °C for 2 days. c, Fold change and statistical significance of
the membrane protein levels in EMC-knockout relative to wild-type cells.
Proteins with a more than 40% decrease in abundance and with P < 0.05 are
highlighted in red. P values were calculated by empirical Bayes t-tests (two-sided)
with no adjustment. d, e, Protein abundance (d) and mRNA levels (e) of nine
putative EMC clients in wild-type and Emc3-knockout (E3KO) yeast strains. The
enhanced GFP (eGFP) reporter is appended to the C termini of the genes. Scale
bar, 3 μm. Data are mean ± s.d. Each black dot indicates the value of a single
independent experiment. Experiments in a–e were repeated three times yielding
similar results.

180°

Emc1

Emc2

Emc5

Emc4

Emc7

Emc1 (^0) Emc1
Emc2
Emc5
Emc4
Emc3
Emc10
Emc6
Lumen
Cytosol
Emc3
100 Å
160
Å
a
180 °
Emc1
Emc2
Emc5
Emc4
Emc7
Emc1 0
Emc1
Emc2
Emc5
Emc4
Emc3
Emc1 0
Emc6 Emc6
Lumen
Cytosol
3
1 2
1
2 3
1
2
1
2
3
1
3 2
1
2
Emc3
b
Emc1 Emc2 Emc3 Emc4 Emc5 Emc6 Emc7 Emc1^0
Cytosol C
N
C
N
N
N
N
C
C
C
C C
C
N
N
N
HH
NTD1
NTD2
23 aa
15 aa
36 aa
16 aa 18 aa
3
1 2 3
2
1
1 2
3 2
1
c
Lumen
Fig. 2 | Structure of the yeast EMC. a, Cryo-EM 3D map of the EMC, showing
front and back views with individual subunits coloured. The dotted black shape
outlines the Emc4 density, which is weaker and partially f lexible (indicated by
the two propagating wave signs). b, An atomic model shown in cartoon and
coloured as in a. Phospholipids and N-glycans are shown in green and red,
respectively. c, Structures of the eight EMC subunits shown separately. aa,
amino acids; HH, horizontal α-helix.