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Supplementary Figure S1:
Digital Video EEG recording of the three patients showing typical hypsarrhythmia in awake state (left column), fragmentation of hypsarrhythmic pattern during sleep
(central column) and epileptic spasms sometimes occurring in clusters (marked by arrows in right column).
Supplementary Figure S2:
Ictal recording of patient IV-1, showing epileptic spasm with typical EEG pattern of slow wave, followed by flattening with posterior fast activity.
Supplementary Figure S3:
Brain MRI images of patient IV-1 at 5 months (A-D) and 2.5 years of age (E-F) , respectively. T2-weighted axial sections, showing normal structure of putamen and striatum (A-C) . T1-weighted sagittal section, showing normal structure of the brain stem and vermis (D) . T2-weighted coronal section, showing mild cortical atrophy and normal basal ganglia (E) . T2-weighted axial section showing mild cortical atrophy (F) .
Supplementary Figure S4:
Sequence alignment of a section of the C-terminal Domain of 539 different GUF1 and
EF4 proteins. This stretch encompasses the A609 residue, which is mutated in the patients affected by isolated West syndrome. The human sequence is repeated at the top and at the bottom of the alignment for reference. The position of the A609S variant is specified by a star (*) and the position of the conserved Q600, I607 and
R610 which three sidechains form a tightly packed motif likely to be important for the proper recognition of the tRNA acceptor stem and D-loop (see main text for details) are indicated by an exclamation mark (!). The Uniprot accession number of each sequence is indicated on the right.
Supplementary Figure S5:
(A) Schematic representation of the pYX232 yeast hGUF1 and hGUF1 A609S expression plasmid used in the complementation assay. (B) Both hGUF1 and hGUF1 A609S overexpression inhibits growth. Wild type (Wt) or the indicated
Δ guf1 strains were grown in medium containing galactose to log phase. Serial 10-fold dilutions were spotted onto agar plates containing 0.5% glycerol and incubated at
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30°C or 37°C as indicated. (C) Expression of hGUF1 and hGUF1 A609S do not modify the level of the Cox2 protein in the Δ guf1 strain. Wild type (Wt) or the indicated Δ guf1 strains were grown to log phase before protein extraction. SDS-PAGE separated proteins were probed with antibodies against the mitochondrially-encoded Cox2 (top lane) or the nuclear genome-encoded Sod1 proteins (bottom lane). (D) Mitochondria of the strains described in (C) were isolated and incubated in the presence of ATP, amino acids and radiolabeled [ 35 S]-methionine for 10 min at either 30 (left panel) or
15 °C (right panel) before visualization of newly synthesized translation products by autoradiography. Visualized bands are annotated on the left, while molecular weight markers are indicated on the right. Note that wild type (Wt), Δguf1 and Δ guf1 overexpressing hGUF1 or hGUF1 A609S strains show similar translation patterns.
Supplementary Figure S6:
(A) Wild type (Wt), oxa1ΔC
or the indicated oxa1ΔC Δguf1 double mutants strains were grown in medium containing galactose to log phase. Serial 10-fold dilutions were spotted onto agar plates containing 2 or 0.5% glycerol and incubated at 30°C
(top panels), 35°C (central panels) or 37°C (bottom panels). (B) oxa1ΔC Δguf1 cells were transformed with plasmids expressing hGuf1 or hGuf1 A609S and harboring a uracil or leucine marker, respectively. Cells were pregrown to log phase in glycerolcontaining medium before inoculation into the competition culture. The latter was repeatedly diluted to promote continuous cell growth. Aliquots were taken after 0, 3,
6, 9 and 12 days, and the proportion of each mutant was determined by plating different volumes on uracil- or leucine-deficient plates. The proportions of cells harboring the hGuf1 A609S plasmids after 0, 3, 6, 9 and 12 days of prolonged growth are shown.
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