SUPPLEMENTAL DATA. Supplementary Methods

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1 SUPPLEMENTL DT Supplementary Methods TP agarose affinity chromatography Peroxisomes extracted from yeast transformed with CTS/pRS416-GPD were resuspended in solubilisation buffer [5 mm Tris-HCl ph 7., 15 mm NaCl, 5 mm MgCl 2, 2% (w/v) Dodecyl Maltoside (DDM), 2% (v/v) glycerol] to a final concentration of 1 mg/ml and incubated for 2 h at ºC. Unsolubilised proteins were removed by centrifugation at 2, g for 15 min. garose beads were added to remove proteins which bind nonspecifically to agarose. Following centrifugation at 2 g for 15 min at 4 C, the supernatants were recovered and mixed with TP agarose beads (N-6 attachment; Sigma, Poole Dorset, UK) preequilibrated in the solubilisation buffer containing 1.4 μm β-mercaptoethanol. The mixture was left to rotate at low speed overnight at 4 C to allow binding of proteins to TP agarose. The mixture was centrifuged at 2 g for 3 min at 4 C and the supernatants were recovered as the unbound fraction. The pellet (beads) was washed three times with solubilisation buffer containing.5% (w/v) DDM. Proteins adsorbed to TP agarose were dissolved in SDS sample buffer containing 1 mm EDT (), or eluted by incubation with 1 or 2 mm TP () and subjected to SDS-PGE followed by immunoblotting for CTS. Subcellular localisation of CTS expressed from CTS/pEL3 Subcellular fractionation was performed as described by Van der Leij et al. (1992) J. Cell iol. 119, Organellar pellets were layered on top of a 15 to 35% (w/v) Nycodenz gradient (12 ml), with a cushion of 1. ml of 5 % (w/v) Nycodenz solution. ll Nycodenz solutions contained 5 mm MES (morpholinoethanesulfonic acid, ph 6.), 1 mm EDT, 1 mm KCl, and 8.5% (w/v) sucrose. The sealed tubes were centrifuged for 2.5 h in a vertical rotor (MSE 8x35) at 19, rpm at 4 C. fter centrifugation, gradients were divided into 12 fractions, which were assayed for activity of marker enzymes. In addition, 15 µl aliquots were taken from the individual fractions derived each from Nycodenz gradient, to which 15 µl of Laemmli sample buffer was added followed by analysis by SDS-PGE and immunoblotting using anti-cts antiserum that recognises the NDs (Fig S1). The activity of the peroxisomal marker 3-hydroxyacyl-Co dehydrogenase was measured on a Cobas-Fara centrifugal analyzer by monitoring the acetoacetyl-co-dependent rate of NDH consumption at 34 nm (van Roermund et al., 28). Fumarase activity was measured on a Cobas-Fara centrifugal analyzer monitoring the PDH production at 365 nm (van Roermund et al., 28). The reaction was started with 1 mm fumarate in an incubation mixture of 1 mm Tris (ph 9.), 1 g/l Triton X-1, 4 U/ml of malate dehydrogenase and 1 mm PD for 5 min at 37 C. Fig. S1. n endogenous protease cleaves CTS. () Varying amounts of peroxisomal protein from CTS-transformed cells were separated by SDS-PGE, and immunoblots were probed with the C- terminal antibody, or with an antibody raised to an N-terminal peptide (Hooks et al., 27). () Schematic representation of the possible origin of the CTS cleavage products as a result of proteolysis in the linker region between the two CTS halves. The C-terminal antibody (epitope indicated by red lines under the green ND domains) detected four bands, migrating close to the 7 kda marker, designated 1-4 in (). Only bands 1 and 3 cross-react with the N-terminal antibody (epitope indicated by red line above the cartoon diagram). This is consistent with bands 1 and 3 being derived from the N-terminal half of CTS, and 2 and 4 from the C-terminal half, since the C-terminal antibody has been shown to recognise both NDs (; De Marcos Lousa et al., 29).Thick and thin arrows indicate major and minor cleavage sites. Fig. S2. Linearity of TPase assay. () The basal TPase activities of peroxisomes from yeast cells transformed with CTS/pRS416-GPD (CTS expressing) or with non-recombinant vector (control) were measured over the protein concentration range of -2 μg. Reactions were incubated at 37º C for 3 min. ll samples were analysed in triplicate, and the experiment was repeated twice and the data were used to compute the means and the standard error of the mean. The plot shows means ± 95% Confidence limit (CL) (computed from SEM for n=6) as indicated by the error bars. () The TPase activities of control and CTS-expressing peroxisomes (1 μg) were measured at 1 min intervals over

2 a 6 min period. Data points were plotted as a scatter plot. The line shows the best fit of the data to a straight line, obtained using linear regression. Fig. S3. Determination of structure-linked latency for CTS. The TPase activity of peroxisomes expressing CTS/pRS416-GPD was determined in the presence of an isotonic buffer (CTS intact and compared to the activity of peroxisomes from control yeast transformed with vector lacking an insert (Control). Peroxisomes from yeast cells expressing CTS were also deliberately ruptured by hypotonic lysis followed by centrifugation at 1 g to collect membranes. The TPase activity of the membranes (CTS ruptured) was determined and compared to the activity of buffered peroxisomes and control. ll samples were analysed in triplicate, and the experiment was repeated twice. The data were used to compute the means and the standard error of mean (SEM). The plot shows mean ± 95% Confidence limit (CL) (computed from SEM for n=6) as indicated by the error bars. Data were analysed by one way NOV and indicated insignificant differences between the TPase activities of the intact and the ruptured peroxisomes (P<.5). Fig. S4. Solubilisation of CTS and analysis of TP binding by TP agarose affinity chromatography. Peroxisomes extracted from yeast expressing CTS/pRS416-GPD were solubilised in the presence of dodecyl maltoside (solubilised CTS) and applied to TP agarose as described in Supplementary Methods. Fractions were separated by SDS-PGE () Immunoblot with anti-cts antiserum showing the unbound material, wash fractions (1-3) and the bound material. () The experiment in was repeated and bound CTS was eluted from the beads with 1 or 2 mm TP. Fig. S5. Subcellular localisation of CTS in pxa1 pxa2δ cells. pxa1 pxa2δ cells transformed with CTS/pEL3 were grown as described in Supplementary Methods. () Homogenate (H), pellet (P) and supernatant (S) fractions were analysed for activity of the peroxisomal marker 3-hydroxyacyl-Co dehydrogenase (HD) and immunoblotted with an anti-cts antiserum. () Organellar pellets were separated on a Nycodenz gradient, as described in Supplementary Methods. Fractions were analysed for 3-hydroxyacyl-Co dehydrogenase and fumarase (mitochondrial marker) and immunoblotted with an anti-cts antiserum.

3 Nyathi et al., Figure S1 C- terminal N-terminal 5 μg 1 μg 2 μg 5 μg 1 μg 2 μg kda Full length CTS 1 Cleaved 3 CTS Proteolytic cleavage TMD1-ND1-TMD2-ND2 and 1 TMD-1ND1 and 2 TMD2-ND2 and 3 TMD1-ND1 and 4 TMD2-ND2

4 Nyathi et al., Figure S C TS expressing inorganic phosphate n mol P i/min C ontrol P eroxisome protein (µg) 12 C TS expressing 1 inorganic phosphate (n mol P i/µg of protein) C ontrol Time (minutes)

5 Nyathi et al., Figure S inorganic phosphate n mol P i/mg/min C T S ruptured C T S intact C ontrol

6 Nyathi et al., Figure S4 Solubilised CTS Unsolubilised CTS Unbound CTS Unbound CTS Wash 1 Wash 2 Wash 3 ound CTS ound CTS 13 CTS 72 CTS* 55 kda 1 mm elution 1 mm bound 2 mm elution 2 mm bound CTS 72 CTS* kda

7 Nyathi et al., Figure S5 % 3-HD activity H P S 13 kda a-cts 6 3-HD fumarase % activity 4 2 a-cts M