Memory (PI)
80
1 hour
(STM)
24 hours
spaced (LTM)
60
60
40
*
40
**
20
20
3
ce
rE
ce 29
r E2
9
/+
ce
rE
CS
ce
rP
ce
rP
/+
9
r E2
ce
CS
0
Supplementary Figure 1 Memory scores of two cer excision strains. The cerE3
excision strain is a revertant, while the cerE29 excision strain shows a LTM-specific
defect. Bars represent means of PIs, error bars are SEM; n = 8 to 10 groups. * and **
indicate significant differences in a T-test (p < 0.05 and p < 0.01, respectively).
a
 ...
 ...
...
...
...
...
...
...
...
...
b
Cathepsin L
1000
1/V
800
x
600
x
x
400
x
x
x
x
6 μM Subs.
4.5 μM Subs.
3 μM Subs.
-20
0
20
40
60
80
100
[Cer] (nM)
Supplementary Figure 2 Cer is a cysteine proteinase inhibitor. a, BLAST alignment
results are presented. “…” indicates incomplete sequence. Cer is highly similar to
cysteine proteinase inhibitors such as murine Cytotoxic T-Lymphocyte Antigen-2 alpha
and beta (CTLA-2 and CTLA-2)1 and Bombyx mori Cysteine proteinase inhibitor
(BCPI)2. Some cysteine proteinases are also shown: Drosophila Cysteine proteinase 1
(CP1) and B. mori cysteine proteinase (BMCP). The degree of similarity is 44%
between Cer and CTLA-2 and 58% between Cer and CP1 if one only takes into
account the CP1 region that aligns with Cer. Drosophila CP1 is a member of the
cathepsin L subfamily3. Sequence analysis suggests that cer arose by partial duplication
of the first three exons of an ancestral CP1 gene4. To our knowledge, no cer mutant has
been previously isolated. Unlike BCPI and CTLA-2, Cer lacks an N-terminal
hydrophobic leader sequence, suggesting that it is a cytoplasmic protein, while BCPI
and CTLA-2 are likely granular or secretory proteins4. b, Cer can inhibit human
cathepsins L. Dixon plot of Cer inhibition of cathepsin L. The Dixon plot is a
convenient way of calculating the Ki of an inhibitor. The reaction velocity is measured
at a fixed concentration of substrate, but at a variety of inhibitor concentrations. A graph
of the reciprocal of velocity against inhibitor concentration is plotted. This is repeated at
different substrate concentrations and a similar line drawn for each concentration of
substrate. A vertical line dropped from the point where the lines intersect each other
down to the inhibitor axis gives -Ki. Calculated Ki = 25 nM.
Cysteine proteinase inhibitors can inhibit different types of cathepsins, due to their three
dimensional structure5. BCPI can inhibit cathepsins L and H but not cathepsin B or
papain6. On the other hand, CTLA-2 inhibits cathepsin L and papain but not cathepsin
B7. Cer inhibits cathepsin L and also cathepsin B (data not shown) but not papain (data
not shown).
Supplementary Figure 3 Confocal microscopy image of a 5122-Gal-4/P(UAS-mCD8GFP) brain. The 5122 line drives the membrane Green Fluorescent Protein into three
different MB lobes: / (yellow) and  (red). The scale bar represents 40 m.
Supplementary reference list:
1. Denizot, F. et al. Novel structures CTLA-2 alpha and CTLA-2 beta expressed in
mouse activated T cells and mast cells and homologous to cysteine proteinase
proregions. Eur J Immunol 19, 631-5 (1989).
2. Yamamoto, N., Hegde, A. N., Chain, D. G. & Schwartz, J. H. Activation and
degradation of the transcription factor C/EBP during long-term facilitation in Aplysia.
J Neurochem 73, 2415-23 (1999).
3. Matsumoto, I., Watanabe, H., Abe, K., Arai, S. & Emori, Y. A putative digestive
cysteine proteinase from Drosophila melanogaster is predominantly expressed in the
embryonic and larval midgut. Eur J Biochem 227, 582-7 (1995).
4. Yamamoto, Y., Kurata, M., Watabe, S., Murakami, R. & Takahashi, S. Y. Novel
cysteine proteinase inhibitors homologous to the proregions of cysteine proteinases.
Curr Protein Pept Sci 3, 231-8 (2002).
5. Turk, D., Podobnik, M., Kuhelj, R., Dolinar, M. & Turk, V. Crystal structures of
human procathepsin B at 3.2 and 3.3 Angstroms resolution reveal an interaction motif
between a papain-like cysteine protease and its propeptide. FEBS Lett 384, 211-4
(1996).
6. Kurata, M. et al. Bombyx cysteine proteinase inhibitor (BCPI) homologous to
propeptide regions of cysteine proteinases is a strong, selective inhibitor of cathepsin
L-like cysteine proteinases. J Biochem (Tokyo) 130, 857-63 (2001).
7. Delaria, K. et al. Inhibition of cathepsin L-like cysteine proteases by cytotoxic Tlymphocyte antigen-2 beta. J Biol Chem 269, 25172-7 (1994).