The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the b-TrCP homologue slimb

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The drosophila Bcl-2 family protein Debcl is targeted to

the proteasome by the b-TrCP homologue slimb

Jessie Colin, Julie Garibal, Amandine Clavier, Aurore Rincheval-Arnold,

Sébastien Gaumer, Bernard Mignotte, Isabelle Guénal, Isabelle Guenal

To cite this version:

homologue Slimb

Article Type: Manuscript

Keywords: Bcl-2 family proteins; Bax; Debcl; proteasome; Slmb; SCF complex

Corresponding Author: Isabelle Guénal

Université de Versailles st Quentin-en-Yvellines Montigny-le-Bretonneux, FRANCE

Corresponding Author Secondary Information:

Corresponding Author's Institution: Université de Versailles st Quentin-en-Yvellines

Corresponding Author's Secondary Institution:

First Author: Jessie Colin, PhD

First Author Secondary Information:

Order of Authors: Jessie Colin, PhD

Julie Garibal, PhD

Amandine Clavier

Aurore Rincheval-Arnold, PhD

Sébastien Gaumer, PhD

Bernard Mignotte, PhD, Professor

Isabelle Guénal



Jessie Colin, Julie Garibal, Amandine Clavier, Aurore Rincheval-Arnold, Sébastien Gaumer, Bernard Mignotte and Isabelle Guénal

Laboratoire de Génétique et Biologie Cellulaire, EA4589, Université de Versailles Saint-Quentin en Yvelines, Ecole Pratique des Hautes Etudes, Bâtiment Simone Veil, 2 avenue de la Source de la Bièvre, 78180 Montigny-le-Bretonneux, France

Dr Isabelle GUENAL

Laboratoire de Génétique et Biologie Cellulaire, EA 4589, Universite de Versailles Saint-Quentin-en-Yvelines, Ecole Pratique des Hautes Etudes,

Bâtiment Simone Veil

2 avenue de la Source de la Bièvre 78180 Montigny-le-Bretonneux

Tel: + 33 (0)1 70 42 94 36 e-mail: isabelle.guenal@uvsq.fr

Manuscript

Click here to download Manuscript: Colin et al 2014 08 27 VF.docx Click here to view linked References

variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of -TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.

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targeting them for UPS-mediated degradation

Only two members of the Bcl-2 family have been identified in Drosophila so far, Buffy and Debcl. Both display a mitochondrial subcellular localization and are apoptotic regulators, suggesting that mitochondria play an important role in apoptosis in Drosophila. Buffy was originally described as an anti-apoptotic Bcl-2 family member (15, 16), but in some cases it can promote cell death (17-20). Debcl (death executioner Bcl 2 homolog), is a multidomain death inducer (20-24) that can be inhibited by direct physical interaction with Buffy (15). When overexpressed in mammalian cells, Debcl induces both cytochrome c release from mitochondria and apoptosis. This protein interacts physically with anti-apoptotic members of the Bcl 2 family, such as Bcl 2 itself, in mammals. Debcl is involved in the control of some developmental cell death processes as well as in the

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Ptc>Debcl-HA,SlmbDN-flag-myc and Ptc>Debcl-HA,Pabp-flag third instar larvae were dissected in PBS

pH 7.6, fixed in PBS/formaldehyde 3.7%, washed three times for 5 min in PBT (PBS, 0.3% Triton). Wing imaginal discs were then dissected and incubated in PBT/FCS (PBS, 0,3% Triton, 10% FCS) at room temperature during 20 min. In situ PLA was performed using the Duolink® kit (Olink Bioscience) essentially according to manufacturer's instructions. Briefly, wing imaginal discs were stained with primary antibodies overnight at 4°C. The antibodies used for Duolink assays and their corresponding dilutions are: anti-HA antibody (ab9110, abcam®, 1/200) and anti-flag.M2 antibody (Agilent, 1/200). After washing, wing imaginal discs were incubated with the secondary oligonucleotide-linked antibodies (PLA probes: anti-Mouse PLUS and anti-Rabbit MINUS) provided in the kit. The oligonucleotides bound to the antibodies were hybridized, ligated, amplified, and detected using a fluorescent probe (Detection Kit 563). Discs were mounted in CitifluorTM (Biovalley) and observed with a Leica SP2 upright confocal microscope.

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To test whether the modulation of Bax- and Debcl-induced cell death involves a direct effect on the level of these proteins, we performed immunostaining and western-blot experiments.

Confocal microscopy was performed on CYMAGES imaging facility.

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Figure 2. overexpression efficiently suppresses bax-induced apoptosis

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Ptc>Debcl-HA,SlmbDN-flag-myc and Ptc>Debcl-HA,Pabp-flag third instar larvae

Figure 1

Figure 3

D

p54

pros25

_skpA

_slmb

_slmb

_morgue

a

0

3.21x10

_1.68x10

2.08x10

-7

1.24x10

7.22x10

D

p54

pros25

_slmb

_morgue

a

2.29x10

_1.69x10

_8.50x10

_0.024

Ws

-6.11

6.965

7.83

2.25

A

B

Figure 7

Supplementary figure 1

In the UY3010 line, P[UY] is inserted in the 5’UTR region of Uba1.

The Drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the



-TrCP homologue Slimb

Jessie Colin, Julie Garibal, Amandine Clavier, Aurore Rincheval-Arnold, Sébastien

Gaumer, Bernard Mignotte and Isabelle Guénal

Supplementary figure 2

Uba1

can be used to overexpress Uba1 under gal4 induction.

(A) Northern blot on total RNA from adult flies. Genotype of flies: y,w

: wild-type;

Uba1

:

Uba1

/CyO

(loss-of-function

allele

of

Uba1),

Uba1

:

Uba1

/CyO; hs>Uba1

: hs-gal4,Uba1

/CyO. Flies were subjected to

heat shock treatment (HS) or not (non-HS). We used probes specific for Uba1 (top)

or rp49 (bottom) as a control. (B) Quantification of Uba1 transcripts. Uba1 transcript

levels were normalized with respect to rp49, with wild-type Uba1 transcript (y,w

)

levels set at 1. The loss-of-function allele of Uba1, Uba1

, had half as many Uba1

Supplementary figure 3

Expression of UAS-debcl in the wing imaginal disc thanks to the ptc-gal4 driver

induces apoptosis along the antero-posterior frontier

The Drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the



-TrCP homologue Slimb

Jessie Colin, Julie Garibal, Amandine Clavier, Aurore Rincheval-Arnold, Sébastien

Gaumer, Bernard Mignotte and Isabelle Guénal

Supplementary figure 4

Effects of mutations on proteasome activity.

Cell lysates from Drosophila head of w

(wi), GMR-Gal4>Uba1

,

GMR-Gal4>skpA

, Dp54 and GMR-Gal4>pros25

flies were migrated on PAGE

and probed with anti-armadillo (N2 7A1 from DSHB, 1/500) and anti-tubulin (E7 from

DSHB, 1/1000) antibodies. Armadillo levels were normalized with tubulin levels and

the ratio was set to 1 for wi flies. Results were from at least 3 independent

experiments per genotype.

Supplementary methods

Heat-shock treatment

Young males were placed in empty tubes at 36°C for 45 minutes. They were

then transferred to a tube containing standard medium and incubated at 21°C for one

day. This temperature shift was repeated 24 hours later. Flies were finally recovered

five hours after heat shock and RNA was extracted.

Northern blot

Total RNA was extracted from 25 males by homogenization in Trizol (Life

Technologies, phenol and guanidine isothiocyanate) and precipitated in isopropanol.

The RNA was resuspended in water and its concentration evaluated by

spectrophotometry (OD 260/280 nm). Northern blotting was then carried out with 30

µg of total RNA, using standard methods. The probes specific for Uba1 or rp49

(ribosomal protein, control for quantification), were generated by PCR, using the

following primers:

5’-GTGTATTCCGACCAGGTTACA-3’ (rp49, forward primer)

5’-ATACAGGCCCAAGATCGTGA-3’ (rp49, reverse primer)

5’-TGTCGTCGCAATTCTATCTCAC-3’ (Uba1, forward primer)

5’-TGGCAATCTGAGAATGATAGCC-3’ (Uba1, reverse primer)

These probes were obtained by random priming and polymerization with the Klenow

fragment of DNA polymerase I. The radioactive nucleotide used was



dCTP

.

Quantification was achieved with a phosphorimager and normalized with respect to

rp49.

TUNEL staining of wing imaginal discs