sec22 regulates er morphology but not autophagy and is ...sec22 regulates er morphology but not...

Sec22 regulates ER morphology but not autophagy in flies

1

Sec22 regulates ER morphology but not autophagy and is required for eye

development in Drosophila

Xiaocui Zhao1#, Huan Yang 1, 2#, Wei Liu1, Xiuying Duan1, Weina Shang1, Dajing

Xia2*, Chao Tong1 *

1 Life Sciences Institute and Innovation Center for Cell Biology, Zhejiang University,

Hangzhou 310058, China

2 School of Public Health, Zhejiang University, Hangzhou 310058, China

Running title: Sec22 regulates ER morphology but not autophagy

# Contributed equally to this work

*Correspondence to: Dr. Chao Tong, Life Sciences Institute, Zhejiang University, 866 Yuhangtang Rd., Hangzhou 310058, China.

Tel: 86-571-88981370; E-mail: [email protected]

Dr. Dajing Xia, School of Public Health, Zhejiang University, 866 Yuhangtang Rd., Hangzhou 310058, China.

E-mail: [email protected]

Keywords: autophagy; Drosophila; Endoplasmic reticulum; Golgi apparatus; SNARE proteins

Background: ER morphological changes are

often observed in disease conditions but the

molecular mechanisms remain unclear.

Results: Malfunction of Sec22 and its binding

partners resulted in ER expansion and defects

in fly eye development.

Conclusion: Sec22 regulates ER morphology

through regulating ER-Golgi trafficking but

not autophagy.

Significance: ER morphology changes under

the disease conditions might due to the defects

of ER-Golgi trafficking.

ABSTRACT

The endoplasmic reticulum (ER) is a highly

dynamic organelle that plays a critical role

in many cellular processes. Abnormal ER

morphology is associated with some human

diseases, although little is known regarding

how ER morphology is regulated. Using a

forward genetic screen to identify genes that

regulated ER morphology in Drosophila, we

identified a mutant of Sec22, the orthologs

of which in yeast, plants, and humans are

required for ER to Golgi trafficking.

However, the physiological function of

Sec22 has not been previously investigated

http://www.jbc.org/cgi/doi/10.1074/jbc.M115.640920The latest version is at JBC Papers in Press. Published on February 10, 2015 as Manuscript M115.640920

Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on April 2, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/cgi/doi/10.1074/jbc.M115.640920

http://www.jbc.org/


2

in animal development. A loss of Sec22

resulted in ER proliferation and expansion,

enlargement of late endosomes, and

abnormal Golgi morphology in mutant

larvae fat body cells. However,

starvation-induced autophagy was not

affected by a loss of Sec22. Mosaic analysis

of the eye revealed that Sec22 was required

for photoreceptor morphogenesis. In Sec22

mutant photoreceptor cells, the ER was

highly expanded, and gradually lost normal

morphology with aging. The rhabdomeres

in mutants were small and sometimes fused

with each other. The morphology of Sec22

mutant eyes resembled the eye morphology

of flies with overexpressed eyeclosed (eyc).

eyc encodes for a Drosophila p47 protein

that is required for membrane fusion. A loss

of Syntaxin5 (Syx5), encoding for a tSNARE

on Golgi, also phenocopied the Sec22

mutant. Sec22 formed complexes with Syx5

and Eyc. Thus, we propose that appropriate

trafficking between the ER and Golgi is

required for maintaining ER morphology

and for Drosophila eye morphogenesis.

The endoplasmic reticulum (ER) plays

pivotal roles in many cellular processes. In

addition to its important function in protein

synthesis, modifications, and quality control,

the ER is also critical for lipid synthesis,

autophagy, and calcium homeostasis (1-3).

The ER makes close contacts with other

organelles, including mitochondria, the Golgi

apparatus, endosomes, peroxisomes, and

lysosomes. It exchanges lipids and proteins

with these organelles to regulate their

biogenesis and function (4, 5). Thus, the ER

has diverse functions and ER malfunctions can

cause numerous human diseases, including

diabetes and neurodegeneration (6).

The ER is a membrane network that

consists of tubules and sheets that extend

throughout a cell (7, 8). The ER is highly

dynamic and constitutively undergoes

rearrangements, which are critical for ER

functions (9-11). ER morphological changes,

including fragmentation and expansion, are

often observed in cells that are subjected to

pathological insults (6). However, the

molecular mechanisms underlying these ER

morphological changes remain unclear.

Using a forward genetic screen to

identify genes that regulated ER morphology,

we obtained a complementation group with

remarkable ER proliferation and expansion.

Molecular mapping located a lesion on Sec22,

a gene encoding for a SNARE protein

enriched on the ER (12, 13). SNARE proteins

are a family of conserved proteins that mediate

membrane fusion between vesicles derived

from one sub-cellular compartment and their

targeted membranes (14, 15). In the secretory

pathway, proteins are transported by vesicle

budding, docking, and fusion. Different

SNAREs mediate the fusion step between

various vesicles and their ultimate destinies.

Vesicle membrane localized v-SNAREs and

targeting membrane localized t-SNAREs form

helix bundles to drive membrane fusion.

Subsequently, these bundled SNAREs are

dissociated and recycled by

N-ethylmaleimide-sensitive factor (NSF) (16).

Sec22 family proteins are highly

conserved from yeast to human. In yeast, there

is only one Sec22 gene and its functions

partially overlap with those of another gene,

ykt6 (13). Sec22p is involved in transport

between the ER and Golgi compartments in

both anterograde and retrograde directions in

yeast (12). In addition, Sec22p is required for

autophagosome biogenesis by regulating the

transport of Atg9, an essential protein for

autophagosome formation, to the phagophore

assembly site (PAS) (17). In both yeast and


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


3

plants, Sec22p/SEC22 regulates cesium

accumulation, which indicates that Sec22

proteins play a role in vacuole function (18). A

loss of SEC22 in plants also results in Golgi

fragmentation and defects in gametophyte

development (19). Studies of the rice fungi

show that Sec22 is required for conidiogenesis,

cell wall integrity, and host plant infection (20).

Several Sec22 genes, SEC22A, SEC22B, and

SEC22C, were also found in humans. In

cultured mammalian cells, SEC22 proteins are

also required for ER to Golgi trafficking (21,

22). However, knocking down SEC22B

expression in cultured mammalian cells does

not cause autophagy defects, presumably due

to redundancy (23). It was recently shown that

SEC22B also has a conserved non-fusogenic

function in plasma membrane expansion (24).

All of these studies demonstrated that

Sec22 was a key regulator of the secretory

pathway. However, surprisingly, there have

been no studies of the physiological functions

of Sec22 in animal development and

morphogenesis. In this study, we found that

Sec22 was an essential gene in flies. A loss of

Sec22 resulted in morphogenesis defects in fly

eyes. The ER was highly proliferated and its

morphology underwent very dramatic changes

in Sec22 mutant flies. In contrast to yeast

orthologs, Sec22 was not required for

starvation-induced autophagy in Drosophila.

Interestingly, a loss of Syntaxin 5 (Syx5),

which encodes for a tSNARE in Golgi, also

phenocopied Sec22 mutants, which indicated

that appropriate trafficking between the ER

and Golgi was critical for ER morphology.

EXPERIMENTAL PROCEDURES

Molecular biology

To generate a Sec22 genomic rescue

construct, a 3.3 kb genomic fragment that

contained the Sec22 gene was amplified using

the primers,

5’-TGAGATCTGTCCATGCAGAGGATGGT

CTTCG-3’ and

5’-TACTCGAGCCAGAGCTTCAGCAGAA

CCACAGC-3’, using genomic DNA that was

purified from FRT19A isogenized flies. To

generate a Sec22 expression vector, we

inserted the Sec22 cDNA into a pattB

UAS-3HA vector. A 3XHA tag was fused to

Sec22 at the N-terminus. To generate a

Flag-tagged Syntaxin 5 expression vector, we

inserted the Flag sequence and Syntaxin 5

cDNA into the pattB UAS expression vector.

The Flag tag was fused to the N-terminus of

the Syx5 protein. A Myc-Eyc expression

vector was generated by inserting full length

eyc cDNA into the pattB UAS-Myc expression

vector. Myc was fused to the N-terminus of the

Eyc protein.

Fly strains

A Sec22 mutant was isolated from an

ethane methylsulfonate (EMS) screen as

previously reported (25). The fly strains

purchased from the Bloomington Drosophila

Stock Center (Indiana University,

Bloomington) were: Df(1)ED6443,

Df(1)BSC534, Dp(1;3)DC012, Dp(1;3)DC013,

P[ey-FLP.N]2; P[EP] Syx5 EP2313P [neoFRT]

40A, P[UASp-GFP.KDEL], and

P[UASp-RFP.KDEL]10. Sec22-genomic-

rescue transgenic flies, UAS-3XHA-Sec22,

UAS-Myc-Eyc, and UAS-Fiag-Syx5

transgenic flies were generated by the Core

Facility of Drosophila Resource and

Technique (Institutes of Biochemistry and Cell

Biology, Shanghai) using standard injection

procedures. Syx5 RNAi fly stains were from

the Tsinghua Fly Stock Center (Beijing,

China).

cDNA analysis of the Sec22 mutant


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


4

Total RNA was isolated from 25 wild

type larvae or larvae with homozygous

mutation in Sec22 using Tirzol reagent

(Invitrogen) according to the manufacturer’s

instructions. cDNAs were synthesized using a

First Strand cDNA Synthesis kit (Invitrogen)

with Oligo dT primers and used in PCR

reactions with the Sec22 -specific primers P1F:

5’-ATGGCGCTGCTGACCATGATAG-3’ and

P1R: 5’

-TTACAGAACCCAGAAATACATG-3’, or

primer P2F:

5’-GTGCTGGCGGGTAACTACTAC-3’ and

P2R:

5’-TTACAGAACCCAGAAATACATG-3’.

The PCR products were sequenced.

Immunostaining for fly fat bodies

Dissected fly fat bodies were fixed

fixed with 3.7% formaldehyde in PBS for 20

min, and then washed 3 times with PBST

(PBS + 0.1% Triton X-100). Tissues were

incubated with a primary antibody (rabbit

anti-HA used at 1:1000; CST) overnight,

extensively washed, and then incubated with a

secondary antibody (Alexa-488 conjugated

used at 1:500; Invitrogen) at room temperature

for 1 h. After extensive washing, samples were

mounted in vector shield and observed by a

confocal microscope.

Phalloidin and immunostaining of the

photoreceptor cells

For the late pupae stage flies, the

dissected brain-eye complexes ware fixed in

PBS with 4% formaldehyde for 20 min. For

the adult flies, heads were fixed in PBS with 4%

formaldehyde for 1 hour upon removal of the

proboscis. Then the photoreceptor cells were

dissected and fixed for 15 min. After the

fixation, the tissues were washed 3 times with

PBST (PBS + 0.4% Triton X-100) and stained

with primary antibody mouse anti-Rh1 (1:50,

Developmental Studies Hybridoma Bank)

overnight at 4 ºC. After extensive washing

with PBST, secondary antibody (1:500,

Invitrogen) and Alexa-488 labeled phalloidin

(1:1000, Invitrogen) were incubated with the

tissues for 2 hours at room temperature.

Samples were washed with PBST for 4 times

and mounted in vector shield and observed by

a confocal microscope.

Cell culture and immunoprecipitation (IP)

S2 cells were cultured at room

temperature and transfected using Opti-PEI.

For Co-IP, cells were harvested at 48 h after

transfection and lysed with lysis buffer. Cell

lysates were incubated with beads (anti-HA,

anti-Myc, or anti-Flag) at 4 ºC for 4 hrs in

lysis buffer. After extensive washing, the IP

products were analyzed by Western blot using

anti-HA (1:1,000), anti-Myc (1:1,000), or

anti-FLAG (1:1,000), followed by an

HRP-conjugated secondary antibody

(1:5,000).

Transmission Electron Microscopy

For electron microscopic observations

of photoreceptors, fly heads were dissected

and fixed in 4% paraformaldehyde and 1%

glutaraldehyde at 4⁰C overnight. Then, the

samples were post-fixed in 2% OsO4 for 2 hrs,

followed by dehydration in ethanol and

propylene oxide. Samples were embedded in

Embed-812 resin (Electron Microscopy

Sciences). TEM images were acquired using a

transmission electron microscope equipped

with a digital camera.

Confocal Microscopy

All mounted samples were examined

and imaged with a confocal microscope

LSM710 (Carl Zeiss) outfitted with a


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


5

Plan-Apochromat 63X 1.4NA oil immersion

objective lens (Carl Zeiss). Data were acquired

using Zen 2010 Software and processed with

Photoshop CS (Adobe).

RESULTS

Identifyinga complementation group, ERM1,

with ER morphology defects.

To identify mutants that had ER

morphology defects, we screened about 700

chemically-induced lethal mutations on X

chromosome (25). We labeled ER structures

using Cg-Gal4 driven UAS-KDEL-GFP

expression in the Drosophila fat body cells and

made mutant clone negatively marked with

RFP (Fig. 1A). By comparing the patterns of

the KDEL-GFP in the RFP negative mutant

cells with those in the neighboring RFP

positive wild type cells, we identified one

complementation group, ERM1. KDEL-GFP

punctate structures became larger and

accumulated inside the ERM1 mutant clones

(Fig. 1B: A’, a’, B’, and b’), which indicated

that the ER was over-proliferated and had

expanded. To determine whether other

organelles were also affected in these mutant

clones, we also examined the patterns for

Rab7-GFP, ManII-GFP (26), and Mito-GFP,

which label late endosomes, the Golgi

apparatus, and mitochondria, respectively.

There was a dramatic enlargement and

accumulation of Rab7-GFP punctate structures

inside ERM1 mutant clones (Fig. 1B: C’, c’,

D’, and d’). The Golgi marker ManII-GFP

became diffusely distributed as compared to its

distribution in a control (Fig. 1B: E’, e’, F’,

and f’). The mitochondria pattern appeared to

be intact inside these mutant clones (Fig. 1B:

G’, g’, H’, and h’). These data suggested that

the ER, late endosomes and Golgi were all

affected in ERM1 mutant cells.

ERM1 mutants correspond to Sec22

mutants

Because the ERM1 phenotypes were

interesting, we performed duplication and

deficiency mapping. ERM1 was rescued by an

X chromosome duplication that covered a

region from 1A1 to 2C1. Several overlapping

deficiencies that covered this region were used

to further narrow down the mutant lesion.

Df(1)BSC534 did but the overlapping

Df(1)ED6433 did not rescue the lethality of

ERM1, which indicated that the mutation was

localized in the region deleted in Df(1)ED6433

but not in Df(1)BSC534. To further pinpoint

the mutant region, small duplications that

covered the deleted region in the deficiencies

were used for a complementation test. ERM1

was rescued by two overlapping small

duplications, Dp(1;3) DC012 and Dp(1;3)

DC013, which narrowed the putative gene

down to nine genes (Fig. 2A). Sequencing

revealed point mutations in Sec22 (Fig. 2B). A

splicing acceptor G to A mutation at the end of

the third intron was detected in ERM1. To

analyze the transcripts in this mutant, we

isolated mRNA from the mutant larvae, made

cDNAs, and sequenced the cDNAs with two

pair of primers. Two different cDNAs were

identified: Sec22 mu1 and Sec22 mu2 (Fig.

2C). An alternative splicing acceptor site was

adopted by Sec22 mu1, which resulted in a

deletion of part of the 4th exon. Wild type

Sec22 encodes for a SNARE protein that

contains a longin domain and a synaptobrevin

domain (Fig. 2D). The frame shift caused by

the deletion in Sec22 mu1 resulted in the

mis-translation of the most part of the

synaptobrevin domain. Sec22 mu2 had an

un-spliced intron and that led to a premature

stop codon in the synaptobrevin domain.

To confirm that ERM1 mutants indeed

corresponded to Sec22 mutants, a genomic


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


6

fragment that contained only the Sec22 gene

was introduced into these mutants (Fig. 2A).

The genomic rescue construct not only fully

rescued the lethality of ERM1 to adulthood but

also rescued the ER morphology defects in the

mutant clones (Fig. 2E). It indicated that Sec22

was the gene affected in this mutant.

Sec22 is not required for autophagy in

Drosophila

A recent study with yeast showed that

Sec22 is required for autophagosome

formation. Because the ER is degraded in the

autophagosome through “ER-phagy” (27), the

accumulated morphologically abnormal ER in

Sec22 mutants could be due to an autophagy

malfunction.

To test if Sec22 was required for

autophagy in flies, we examined different

autophagy markers in Sec22 mutant cells after

their starvation. We labeled autophagy

vacuoles in fat bodies by expressing different

markers driven by a fat body Gal4 driver,

Cg-Gal4 (28). We negatively labeled the Sec22

mutant clones with RFP in third instar larvae

fat bodies, and then examined the different

autophagy markers after starvation for 4 hours.

Autophagy initiation requires type III

PI3K activity, which can be monitored based

on the intracellular distribution of FYVE-GFP.

Without PI3K activity, FYVE-GFP signals are

diffusely distributed inside cells. If PI3K is

activated, then FYVE-GFP exhibits punctate

staining (29). In Sec22 mutant cells,

FYVE-GFP exhibited a punctate distribution

that was similar to that in control cells (Fig. 3

A, A’), which indicated that PI3K activity was

intact.

Atg8 is an ubiquitin-like protein that is

required for autophagosome formation and

autophagy progression (30). Without

starvation, Atg8 signals are diffusely

distributed inside cells. Upon starvation, Atg8

forms punctate structures and labels

autophagosomes. Once autophagosomes fuse

with lysosomes, Atg8 is degraded inside

autolysosomes. In Sec22 mutant clones,

Atg8-GFP punctate structures were formed

and these patterns were comparable to those in

controls’ (Fig. 3B, B’), which indicated that

Sec22 was not required for autophagosome

formation.

To monitor autophagosome and

lysosome fusion, Atg8-GFPRFP was used as a

marker. GFP signals but not RFP signals are

quenched inside acidic autolysosomes; thus,

autophagosomes will appear yellow and

autolysosomes will appear red (31). If there

are fusion defects between autophagosomes

and lysosomes, then most Atg8 punctate

structures will appear as yellow; otherwise, the

majority of Atg8 punctate structures will

appear as red. The yellow and red signals of

Atg8-GFPRFP punctate structures in Sec22

mutant clones were same as those in control

cells (Fig. 3C, C’), which indicated that Sec22

was not required for autophagosome

maturation.

To examine autophagy flux in Sec22

mutant cells, we checked the patterns of p62

(32), an adapter protein that is digested via

autophagy. There was no difference in the p62

protein distribution between Sec22 mutants

and wild type controls’ (Fig. 3D, D’).

In summary, Sec22 was not required

for autophagy in fly fat body cells. Thus, the

accumulated ER and morphological changes

were not due to autophagy failure.

Syx5 interacts with Sec22 to regulate ER

morphology

Because autophagy was not affected


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


7

by a loss of Sec22, the accumulation of

morphologically abnormal ER could have

resulted from other conserved functions of

Sec22. In yeast, Sec22 forms a complex with

the Golgi SNARE protein Sed5p to mediate

the fusion of ER-derived vesicles with the

Golgi compartment (33). In Sec22 mutant fat

body cells, the ER was expanded and Golgi

morphology had changed, which indicated that

Sec22 indeed was required for membrane

trafficking between the ER and Golgi in flies.

Thus, we hypothesized that trafficking defects

between the ER and Golgi had resulted in the

ER morphological changes.

To test this, we first examined

whether Syntaxin 5 (Syx5), encoded by the fly

ortholog of sed5, could bind to Sec22. We

performed immunoprecipitation (IP) using

cultured S2 cells and found that Sec22 indeed

formed a complex with Syx5 (Fig. 4A). When

we knocked down Syx5 in fly fat bodies using

RNAi, we found that KDEL-RFP punctate

structures became larger and had accumulated

inside cells (Fig. 4B, C). This indicated that

the ER had expanded in Syx5 RNAi tissues, a

phenotype that was very similar to the Sec22

mutants. Sec22 is a v-SNARE on the ER

membrane and Syx5 is a t-SNARE on the

Golgi compartment. The ER morphology

change in those cells that had lost either

SNARE protein suggested that the trafficking

failure between the ER and Golgi had

triggered ER expansion.

Interestingly, Syx5 overexpression in fat body

cells also led to ER expansion similar to the

ER morphology in the Syx5 RNAi tissues,

suggesting Syx5 overexpression has dominant

negative effects (Fig. 4E). In yeast, ER-Golgi

transport requires four SNARE proteins,

Sec22p, Bos1p, Bet1p, and Sed5p (encoded by

a yeast ortholog of Syx5), to form complexes.

The dominant negative effects of Syx5

overexpression probably is due to the extra

Syx5 competing with the endogenous Syx5 to

form ineffective complexes with other

endogenous SNARE proteins, such as Sec22

or the proteins encoded by the orthologs of

Bos1p or Bet1p. Strikingly, when we

co-expressed Sec22 and Syx5, the ER

punctuate structures in the fat body cells were

much larger than those in the cells with Syx5

overexpression alone (Fig. 4D-F). Since Sec22

overexpression did not have ER morphology

defects, the enhancement of the Syx5

overexpression phenotypes suggested that

Sec22 might form a complex with Syx5 and

together they were a better competitor than

Syx5 alone to titrate out of other SNARE

proteins. These data implied that Sec22 and

Syx5 might function in a same pathway to

regulate ER morphology.

Sec22 is required for eye morphogenesis

The dramatic ER morphological

changes in the Sec22 mutant cells suggested

that Sec22 played a critical role in regulating

ER function. However, the physiological

function of Sec22 has not been previously

investigated in animals. We found that a Sec22

hemizygous mutant cannot survive to the

adulthood. To investigate whether Sec22 was

required for nerve system development and

neuron homeostasis, we generated Sec22

mutant clones in the eye using the eyFLP

system. In wild type control eyes, round,

tightly packed and oval shaped rhabdomeres

were in a trapezoid arrangement. In Sec22

mutant eyes, irregularly shaped rhabdomeres

were small and sometimes fused with each

other (Fig. 5A, a, C and c). Inside

photoreceptor cells, the ER was highly

expanded with numerous stacks and gradually

lost its normal morphology with aging (Fig.

5A-d). Occasionally, lipid droplets could be

detected in the cytoplasm, another hallmark of


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


8

ER malfunction (Fig. 5A-d).

It has been reported that mis-trafficking of

Rhodopsin 1 (Rh1) leads to retinal

developmental defects and degeneration (34,

35). Rh1 is a light sensor that presents in the

R1-R6 photoreceptor cells. During fly

photoreceptor cell development, Rh1 is

synthesized and matured in the ER and

transport to the rhabdomeres through the Golgi.

The ER defects are likely to cause

mis-trafficking of Rh1 (34, 35). We therefore

tested whether there is Rh1 trafficking defects

in the Sec22 mutants. We made Sec22 mutant

clones in the fly eyes and stained the tissue

with anti-Rh1 antibody and Phalloidin to

examine the distribution Rh1 in the

photoreceptor cells at the late pupae (84h APF)

and adult stages. There was a massive

accumulation of Rh1 in the cytoplasm (Fig.

5E), which might explain the degeneration

phenotypes we observed in Sec22 mutant eyes.

Syx5 mutants, Sec22 mutants, and Eyc

overexpression animals all have similar ER

phenotypes

Studies of Syx5 functions in the fat

body cells suggested that it might form a

complex with Sec22 to regulate ER

morphology. We then tested whether Syx5

mutants also had phenotypes similar to those

of Sec22 mutants in the eyes. Indeed, the

rhabdomeres were small and irregularly

shaped in the Syx5 mutants. The photoreceptor

cells were also packed with over-proliferated

ER and lipid droplets (Fig. 6A, a).

The photoreceptor morphogenesis

defects and ER accumulation in Sec22 mutants

were reminiscent of a mutant called eyes

closed (eyc) (36). eyc encodes for a fly p47

protein that forms a complex with p97 ATPase

to regulate membrane fusion (37). Eyc

overexpression during the early pupal stage

resulted in fused rhabdomeres. Eyc

mis-expression at a later stage resulted in ER

proliferation in the photoreceptor cells (Fig.

6B, b) (36). The trafficking of Rh1 is defective

in the fly eyes with Eyc overexpressed (36).

All of these phenotypes were very similar to

what we observed in Sec22 mutant eyes. To

test whether Sec22 and Eyc interacts with each

other, we performed IP experiments with

cultured fly S2 cells. Indeed, Sec22 bound to

Eyc (Fig. 6C, D). We wondered whether Sec22,

Syx5, and Eyc function in a same pathway to

regulate ER morphology and photoreceptor

cell morphogenesis. We knocked Syx5 known

or overexpressed Eyc in the Sec22 mutant

background using MARCM technology and

analyzed the ER morphology in the fat body

cells. Both Syx5 RNAi and Eyc overexpression

in the Sec22 mutant cells led to cell growth

defects. The cells became very small, which

hindered the ER morphology analysis (Fig. 6

E-J). The synergistic effects of regulating cell

size suggested these proteins may have

paralleled functions in cell growth. But

whether they work together in regulating ER

morphology is still an open question.

Since both Eyc and Syx5 interact with

Sec22, we hypothesized that Eyc might

compete with Syx5 to bind Sec22. However,

we did not observe any decrease of the

interaction between Sec22 and Syx5 when Eyc

was overexpressed (Fig. 6K). We also did not

observe any alleviation of ER morphology

defects in Eyc overexpression cells when

Sec22 was overexpressed. Therefore, Eyc

overexpression phenotypes were not due to

competing Sec22 with Syx5 (Fig. 6L-O).

DISCUSSION


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


9

Losses of Sec22 and Syx5 result in ER

expansion

The ER is highly dynamic and its

morphology is tightly regulated (10, 11).

Abnormal ER morphology is associated with

hereditary spastic paraplegias (HSP) and

related disorders (38). In this study, we found

that both Sec22 and Syx5 mutants had an

over-proliferated ER and expanded ER lumens.

Sec22 is primarily localized on the ER and

Syx5 is found on the Golgi. The interaction

between Sec22 and Syx5 mediates the fusion

of ER-derived vesicles to Golgi cysts. The

similar ER phenotypes between Sec22 and

Syx5 mutants suggested that defective

ER-Golgi trafficking resulted in ER

proliferation and morphology defects.

ER proliferation has been found in

cells that were exposed to multiple

pathological insults; however, the molecular

mechanisms involved are less well understood

(6). It would be interesting to test whether the

trafficking route between the ER and Golgi is

blocked in these disease conditions. It is still

not clear how these trafficking defects result in

ER proliferation and morphology defects. ER

stress could result in ER proliferation. Thus, it

needs to be determined whether these Sec22

mutants have ER stress responses.

Eyc, p97 and ER dynamics

SNARE proteins can assemble into

unproductive cis-SNARE complexes that need

to be dissociated by two ATPases: NSF

(N-ethylmaleimide-sensitive factor) (39) and

p97 (40-42). After their dissociation, SNAREs

are available to form trans-complexes and

promote another round of fusion. p47 is an

adaptor protein that mediates the interaction

between p97 and a SNARE complex (43, 44).

Interestingly, overexpression of Eyc (fly

ortholog of p47) mimicked the loss of Sec22

and Syx5, which suggested that Eyc functioned

within a paralleled or a same pathway as

Sec22 and Syx5. We found that Sec22 could

bind to Eyc. However, Eyc overexpression

phenotypes are not due to Eyc competing

Sec22 with Syx5. It is possible that the excess

Eyc interferes the organization of the whole

SNARE complex required for ER-Golgi

trafficking. It is also possible that Eyc works

in a paralleled pathway independent of Sec22

to regulate ER morphology.

Sec22 and autophagy

Autophagy is a critical degradative

pathway that is required for cellular

homeostasis (45). SNAREs are important for

membrane homophilic fusion during

autophagosome formation (17, 46). Sec22

facilitates Atg9 recruitment to the PAS and,

therefore, is required for autophagosome

formation in yeast (17). However, in our study,

Sec22 was dispensable for starvation-induced

autophagy in flies. In yeast, autophagosomes

are generated from the PAS, which has not yet

been identified in fly and mammalian cells

(31). It is possible that different autophagic

initiation structures determine whether Sec22

is required for autophagy. Interestingly, no

autophagy defects are observed when SEC22B

is knocked down in mammalian cells (46).

In yeast, ykt6 function overlaps with

that of Sec22 (13). Ykt6p is required for

autophagosome formation in yeast (17), and

Ykt6p is highly conserved. It is possible that

Ykt6 and Sec22 have redundant roles during

autophagy.

The ER is one of the membrane

sources of the autophagosome (47). In Sec22

mutants, the ER was highly proliferated. The

lumens of the ER were expanded and ER


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


10

morphology changed dramatically. However,

autophagosome formation was not affected in

Sec22 mutants, which indicated that

appropriate ER morphology was not

absolutely required for autophagosome

formation.

In summary, we found that trafficking

between the ER and Golgi mediated by Sec22

and Syx5 was critical for maintaining ER

morphology. Sec22 was also required for

development and eye morphogenesis in flies.

ACKNOWLEGEMENT

We thank Dr. Hugo Bellen, Bloomington

Drosophila Stock Center, and the

Developmental Studies Hybridoma Bank for

providing reagents. C. Tong was supported by

the National Basic Research Program of China

(2012CB966600, 2014CB943100), the

National Natural Science Foundation of China

(31271432), and Specialized Research Fund

for the Doctoral Program of Higher Education

(20130101110116)

REFERENCES

1. Friedman, J. R., and Voeltz, G. K. (2011) The ER in 3D: a multifunctional dynamic

membrane network. Trends Cell Biol 21, 709-717

2. Ellgaard, L., and Helenius, A. (2003) Quality control in the endoplasmic reticulum. Nat Rev

Mol Cell Biol 4, 181-191

3. Lamb, C. A., Yoshimori, T., and Tooze, S. A. (2013) The autophagosome: origins unknown,

biogenesis complex. Nat Rev Mol Cell Biol 14, 759-774

4. Rowland, A. A., and Voeltz, G. K. (2012) Endoplasmic reticulum-mitochondria contacts:

function of the junction. Nat Rev Mol Cell Biol 13, 607-625

5. Brandizzi, F., and Barlowe, C. (2013) Organization of the ER-Golgi interface for membrane

traffic control. Nat Rev Mol Cell Biol 14, 382-392

6. Hetz, C., and Mollereau, B. (2014) Disturbance of endoplasmic reticulum proteostasis in

neurodegenerative diseases. Nat Rev Neurosci 15, 233-249

7. Voeltz, G. K., and Prinz, W. A. (2007) Sheets, ribbons and tubules - how organelles get their

shape. Nat Rev Mol Cell Biol 8, 258-264

8. Mattaj, I. W. (2004) Sorting out the nuclear envelope from the endoplasmic reticulum. Nat

Rev Mol Cell Biol 5, 65-69

9. Vedrenne, C., and Hauri, H. P. (2006) Morphogenesis of the endoplasmic reticulum: beyond

active membrane expansion. Traffic 7, 639-646

10. Chen, S., Novick, P., and Ferro-Novick, S. (2013) ER structure and function. Curr Opin Cell

Biol 25, 428-433

11. Lin, S., Sun, S., and Hu, J. (2012) Molecular basis for sculpting the endoplasmic reticulum

membrane. Int J Biochem Cell Biol 44, 1436-1443

12. Lewis, M. J., Rayner, J. C., and Pelham, H. R. (1997) A novel SNARE complex implicated in

vesicle fusion with the endoplasmic reticulum. EMBO J 16, 3017-3024

13. Liu, Y., and Barlowe, C. (2002) Analysis of Sec22p in endoplasmic reticulum/Golgi transport

reveals cellular redundancy in SNARE protein function. Mol Biol Cell 13, 3314-3324


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


11

14. Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) A

protein assembly-disassembly pathway in vitro that may correspond to sequential steps of

synaptic vesicle docking, activation, and fusion. Cell 75, 409-418

15. Sollner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst,

P., and Rothman, J. E. (1993) SNAP receptors implicated in vesicle targeting and fusion.

Nature 362, 318-324

16. Rizo, J., and Sudhof, T. C. (2012) The membrane fusion enigma: SNAREs, Sec1/Munc18

proteins, and their accomplices--guilty as charged? Annu Rev Cell Dev Biol 28, 279-308

17. Nair, U., Jotwani, A., Geng, J., Gammoh, N., Richerson, D., Yen, W. L., Griffith, J., Nag, S.,

Wang, K., Moss, T., Baba, M., McNew, J. A., Jiang, X., Reggiori, F., Melia, T. J., and

Klionsky, D. J. (2011) SNARE proteins are required for macroautophagy. Cell 146, 290-302

18. Draxl, S., Muller, J., Li, W. B., Michalke, B., Scherb, H., Hense, B. A., Tschiersch, J., Kanter,

U., and Schaffner, A. R. (2013) Caesium accumulation in yeast and plants is selectively

repressed by loss of the SNARE Sec22p/SEC22. Nat Commun 4, 2092

19. El-Kasmi, F., Pacher, T., Strompen, G., Stierhof, Y. D., Muller, L. M., Koncz, C., Mayer, U.,

and Jurgens, G. (2011) Arabidopsis SNARE protein SEC22 is essential for gametophyte

development and maintenance of Golgi-stack integrity. Plant J 66, 268-279

20. Song, W., Dou, X., Qi, Z., Wang, Q., Zhang, X., Zhang, H., Guo, M., Dong, S., Zhang, Z.,

Wang, P., and Zheng, X. (2010) R-SNARE homolog MoSec22 is required for conidiogenesis,

cell wall integrity, and pathogenesis of Magnaporthe oryzae. PLoS One 5, e13193

21. Paek, I., Orci, L., Ravazzola, M., Erdjument-Bromage, H., Amherdt, M., Tempst, P., Sollner,

T. H., and Rothman, J. E. (1997) ERS-24, a mammalian v-SNARE implicated in vesicle

traffic between the ER and the Golgi. J Cell Biol 137, 1017-1028

22. Hay, J. C., Chao, D. S., Kuo, C. S., and Scheller, R. H. (1997) Protein interactions regulating

vesicle transport between the endoplasmic reticulum and Golgi apparatus in mammalian cells.

Cell 89, 149-158

23. Moreau, K., Ravikumar, B., Renna, M., Puri, C., and Rubinsztein, D. C. (2011)

Autophagosome precursor maturation requires homotypic fusion. Cell 146, 303-317

24. Petkovic, M., Jemaiel, A., Daste, F., Specht, C. G., Izeddin, I., Vorkel, D., Verbavatz, J. M.,

Darzacq, X., Triller, A., Pfenninger, K. H., Tareste, D., Jackson, C. L., and Galli, T. (2014)

The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion. Nat Cell

Biol 16, 434-444

25. Zhang, K., Li, Z., Jaiswal, M., Bayat, V., Xiong, B., Sandoval, H., Charng, W. L., David, G.,

Haueter, C., Yamamoto, S., Graham, B. H., and Bellen, H. J. (2013) The C8ORF38

homologue Sicily is a cytosolic chaperone for a mitochondrial complex I subunit. J Cell Biol

200, 807-820

26. Ye, B., Zhang, Y., Song, W., Younger, S. H., Jan, L. Y., and Jan, Y. N. (2007) Growing

dendrites and axons differ in their reliance on the secretory pathway. Cell 130, 717-729

27. Bernales, S., Schuck, S., and Walter, P. (2007) ER-phagy: selective autophagy of the

endoplasmic reticulum. Autophagy 3, 285-287

28. Juhasz, G., and Neufeld, T. P. (2008) Experimental control and characterization of autophagy

in Drosophila. Methods Mol Biol 445, 125-133

29. Juhasz, G., Hill, J. H., Yan, Y., Sass, M., Baehrecke, E. H., Backer, J. M., and Neufeld, T. P.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


12

(2008) The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR

signaling in Drosophila. J Cell Biol 181, 655-666

30. Scott, R. C., Schuldiner, O., and Neufeld, T. P. (2004) Role and regulation of

starvation-induced autophagy in the Drosophila fat body. Dev Cell 7, 167-178

31. Mizushima, N., Yoshimori, T., and Levine, B. (2010) Methods in mammalian autophagy

research. Cell 140, 313-326

32. Pircs, K., Nagy, P., Varga, A., Venkei, Z., Erdi, B., Hegedus, K., and Juhasz, G. (2012)

Advantages and limitations of different p62-based assays for estimating autophagic activity in

Drosophila. PLoS One 7, e44214

33. Sacher, M., Stone, S., and Ferro-Novick, S. (1997) The synaptobrevin-related domains of

Bos1p and Sec22p bind to the syntaxin-like region of Sed5p. J Biol Chem 272, 17134-17138

34. Colley, N. J., Cassill, J. A., Baker, E. K., and Zuker, C. S. (1995) Defective intracellular

transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration. Proc

Natl Acad Sci U S A 92, 3070-3074

35. Kumar, J. P., and Ready, D. F. (1995) Rhodopsin plays an essential structural role in

Drosophila photoreceptor development. Development 121, 4359-4370

36. Sang, T. K., and Ready, D. F. (2002) Eyes closed, a Drosophila p47 homolog, is essential for

photoreceptor morphogenesis. Development 129, 143-154

37. Uchiyama, K., and Kondo, H. (2005) p97/p47-Mediated biogenesis of Golgi and ER. J

Biochem 137, 115-119

38. Fink, J. K. (2013) Hereditary spastic paraplegia: clinico-pathologic features and emerging

molecular mechanisms. Acta Neuropathol 126, 307-328

39. Block, M. R., Glick, B. S., Wilcox, C. A., Wieland, F. T., and Rothman, J. E. (1988)

Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport. Proc

Natl Acad Sci U S A 85, 7852-7856

40. Acharya, U., Jacobs, R., Peters, J. M., Watson, N., Farquhar, M. G., and Malhotra, V. (1995)

The formation of Golgi stacks from vesiculated Golgi membranes requires two distinct fusion

events. Cell 82, 895-904

41. Latterich, M., Frohlich, K. U., and Schekman, R. (1995) Membrane fusion and the cell cycle:

Cdc48p participates in the fusion of ER membranes. Cell 82, 885-893

42. Rabouille, C., Levine, T. P., Peters, J. M., and Warren, G. (1995) An NSF-like ATPase, p97,

and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell 82, 905-914

43. Edwardson, J. M. (1998) Membrane fusion: all done with SNAREpins? Curr Biol 8,

R390-393

44. Mellman, I. (1995) Enigma variations: protein mediators of membrane fusion. Cell 82,

869-872

45. Levine, B., and Kroemer, G. (2008) Autophagy in the pathogenesis of disease. Cell 132,

27-42

46. Moreau, K., and Rubinsztein, D. C. (2012) The plasma membrane as a control center for

autophagy. Autophagy 8, 861-863

47. Bernard, A., and Klionsky, D. J. (2013) Autophagosome formation: tracing the source. Dev

Cell 25, 116-117


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


13

FIGURE LEGENDS

Figure 1. Identifying a complementation group with ER morphology defects

(A) . Screening strategy used to identify mutants with ER morphology defects. (B). Complementation

group ERM1 had an expanded ER, enlargements of late endosomes, and abnormal Golgi

morphology. However, mitochondria were intact. CTL and mutant clones are marked by a loss of

RFP (red) and outlined by the dashed lines. (a’-h’) Green channels of (A’-H’). KDEL-GFP was

used to label the ER, Rab7-GFP was used to label late endosomes, ManII-GFP was used to label

the Golgi apparatus, and Mito-GFP was used to label mitochondria.

Figure 2. ERM1 mutants correspond to Sec22 mutants.

(A) Deficiency and duplication mapping located the ERM1 lesion to a small genomic fragment that

contains 9 genes, including Sec22. (B). Sanger sequencing reviewed a G to A mutation at the

splicing acceptor site on Sec22. (C). Diagram indicating the genomic organization and the

resulting mRNA of the Sec22 locus. The exons were shown as bars with different colors and the

introns were shown as broken lines. The positions of the primers used for RT-PCR were indicated.

The point mutation on ERM1 resulted in two transcripts (Sec22 mu1 and Sec22 mu2) different

from the wild type (Sec22 WT). The alignment of the Sec22 mu1 with Sec22 WT showed that a

deletion in Sec22 mu1 resulted from an alternative splicing site used by Sec22 mu1. The

alignment of Sec22 mu2 with Sec22 WT showed that an intron was failed to be spliced and an

insertion was produced in Sec22 mu2. (D). Diagram indicating the domain organization of Sec22

protein. The proteins encoded by Sec22 WT, mu1, and mu2 were shown. (E). Sec22 genomic

rescue construct rescued the ER morphology defects of Sec22 mutant clones. The ER was labeled

with KDEL-GFP. The clones were negatively labeled with RFP.

Figure 3. Sec22 is not required for starvation-induced autophagy.

No obvious autophagy defects could be detected in Sec22 mutant clones (absence of RFP: red in

A, A’, C, C’, D, D’). (A, A’). FYVE-GFP indicated that PI3K activity, whose activity is

required for autophagy initiation, was intact in Sec22 mutant cells. (B, B’). Upon starvation,

Atg8-GFP formed punctate structures inside fat body cells. Both control (CTL) and Sec22 clones

had similar responses to starvation. (C, C’). MARCM clones of CTL and Sec22 mutant cells

showed the Atg8-GFPRFP signals for monitoring autophagosome and lysosome fusion. There

was no difference between CTL and Sec22 cells. (D, D’). Distribution of p62, an autophagy

substrate, in Sec22 mutant cells was similar to that in CTL cells.

Figure 4. Syx5 interacts with Sec22 to regulate ER morphology

(A) Flag-tagged Syx5 pulled down HA-tagged Sec22 (top two panels). HA-tagged Sec22 also pulled

down Flag-tagged Syx5 (bottom two panels). (B-F). Syx5 RNAi (C) and overexpression (E)

resulted in KDEL-RFP labeled ER proliferation and expansion. Sec22 overexpression alone had

no effect on the ER morphology (D), but enhanced the ER morphology defects when

co-expressed with Syx5 (F). Cg-Gal4 was used to drive the expression of KDEL-RFP,

Syx5shRNA, UAS-Flag-Syx5, and UAS-3XHA Sec22.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


14

Figure 5. Sec22 is required for eye morphogenesis

(A, a, C, and c). In one-day-old wild type control (CTL) eyes, round, tightly packed, oval shaped

rhabdomeres were in a trapezoid arrangement (A, a). In age-matched Sec22 mutant eyes (C, c),

the rhabdomeres were small and sometimes fused with each other (blue arrows). The ER was

highly proliferated (red arrows) and lipid droplets (yellow arrows) were accumulated. (B, b, D, d).

The ER gradually lost its shape and became highly accumulated inside photoreceptor (PR) cells

with aging. (E).The Rh1 trafficking was defective in Sec22 mutant PR cells. The Sec22 mutant

clones were generated by eyFLP/FRT system. The mutant area was outlined and the typical

mutant PR cells were pointed with yellow arrows. Pha (Phalloidin, red), Rh1 (blue).

Figure 6. Syx5 mutants, Sec22 mutants, and Eyc overexpression animals have similar ER

phenotypes

(A, a). One-day-old Syx5 mutants had phenotypes similar to those of Sec22 mutants. Rhabdomeres

were small and fused (blue arrows), ER was proliferated (red arrows) and lipid droplets were

accumulated (yellow arrows). (B, b). GMR-Gal4 driven Eyc overexpression resulted in ER

proliferation (red arrows) and lipid droplets accumulation (yellow arrows). (C). HA-tagged

Sec22 pulled down Myc-tagged Eyc. (D). Myc-tagged Eyc also pulled down HA-tagged Sec22.

(E-J). Syx5 RNAi and Eyc overexpression in the Sec22 mutant cells led to cell growth defects.

ER was labeled with KDEL-RFP (red). (E). Control clone (CTL). (F). Sec22 mutant clone. (G).

Syx5 RNAi clone. (H).Syx5 shRNA expressed in Sec22 mutant clone. (I). Eyc overexpressed

clone. (J). Eyc overexpressed in Sec22 mutant clone. (K). Eyc overexpression cannot interfere

with complex formation between Syx5 and Sec22. S2 cells with indicated protein expressed.

HA-tagged Sec22 was immunoprecipated with anti-HA anibody and the amount of Flag-tagged

Syx5 was detected with anti-Flag antibody. Eyc expression did not reduce the amount of Syx5

interacted with Sec22. (L-O). Sec22 overexpression cannot rescue the ER morphology defects

caused by Eyc overexpression. ER was labeled with KDEL-RFP (red). (L). Control. (M).

Cg-Gal4 driven UAS-3HA-Sec22 overexpression. (N). Cg-Gal4 driven UAS-Myc-Eyc

overexpression. (O). Cg-Gal4 driven both UAS-3HA-Sec22 and UAS-Myc-Eyc expression.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

TongXiaocui Zhao, Huan Yang, Wei Liu, Xiuying Duan, Weina Shang, Dajing Xia and Chao

development in DrosophilaSec22 regulates ER morphology but not autophagy and is required for eye

published online February 10, 2015J. Biol. Chem.

10.1074/jbc.M115.640920Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M115.640920

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M115.640920v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2015/02/10/jbc.M115.640920

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2015/02/10/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2015/02/10/jbc.M115.640920

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

sec22 regulates er morphology but not autophagy and is ...sec22 regulates er morphology but not...

Documents