Figure 2
Cytosolic re-localization of the isobutanol synthesis enzyms Ilv2, Ilv5 and Ilv3. (A) N-terminal amino acid sequences of the precursor proteins Ilv2, Ilv5 and Ilv3. The N-termini of enzymes Ilv2, Ilv5 and Ilv3 were shortened to eliminate the N-terminal import signal sequences. Different truncations were constructed which were based on alignments with bacterial homologues and are indicated by inverted triangles. Truncations predicted by Mitoprot analysis are indicated by arrows. (B-D) Western blot analyses of wild-type and N-terminally truncated Ilv2, Ilv5 and Ilv3 proteins carrying a C-terminal 6His-tag. CEN.PK2-1C cells containing overexpression plasmids for the different proteins were grown on selective SCD media into the exponential growth phase, crude extracts were prepared and subjected to Western blot analyses. Bands of interest are framed. Panel B: Lane1: Ilv2ΔN54; Lane2: Ilv2ΔN85; Lane3: wild-type Ilv2. Panel C: Lane1: Ilv5ΔN48; Lane2: wild-type Ilv5. Panel D: Lane1: Ilv3ΔN19; Lane2: Ilv3ΔN34; Lane3: Ilv3ΔN42; Lane4: Ilv3ΔN50; Lane5: Ilv3ΔN19DE; Lane6: wild-type Ilv3. (E-G) Indirect immunofluorescence microscopy of wild-type and N-terminally truncated proteins carrying a C-terminal 6His-tag. Yeast cells as under B-D were grown on selective SCD media into the exponential growth phase, harvested and prepared as described in Material and Methods. α-His antibodies were applied for the visualisation of Ilv enzymes, α-Hsp70 antibodies for cytosolic staining. Panel E: localization of Ilv2 variants. Panel F: localization of Ilv5 variants and empty vector (ev). Panel G: localization of Ilv3 variants.