Volume 8 issue 6 June 2007
The peroxin PEX14 of Neurospora crassa is essential for the biogenesis of both glyoxysomes
and Woronin bodies
David Managadze, Christian Würtz, Martin Sichting, Gerd Niehaus, Marten Veenhuis, and Hanspeter Rottensteiner

Supplementary Table I: Plasmids and Oligonucleotides used

Table 1 (doc)

Figure 1. Alignment of PEX13 with yeast orthologs. PEX13 sequences from Neurospora crassa (NCU02618.3; NcPEX13), Pichia pastoris (Q92266; PpPEX13), and Saccharomyces cerevisiae (YLR191w; ScPEX13) were used to perform a multiple sequence alignment using CLUSTALW with its default parameters (http://www.ebi.ac.uk/clustalw/). Identical amino acids are shaded in black and similar ones in gray using BoxShade (http://www.ch.embnet.org/software/BOX_form.html).

Figure 1 (doc)

Figure 2. Alignment of PEX14 with yeast orthologs. PEX-14 sequences from Neurospora crassa (NCU03901.3; NcPEX14), Pichia pastoris (AAG28574; PpPEX14), and Saccharomyces cerevisiae (YGL153w; ScPEX14) were used to perform a multiple sequence alignment using CLUSTALW with its default parameters. Identical amino acids are shaded in black and similar ones in gray using BoxShade.

 Figure 2 (doc)

Figure 3. Specificity of antibodies generated against PEX14 and PEX13.
(A, B) PEX14 antibody test. Antisera generated against a recombinant GST fusion of N. crassa PEX14 were tested for specificity using whole-cell extracts prepared from the oleic acid-induced S. cerevisiae wild-type strain UTL-7A, an otherwise isogenic pex14Δ strain, and the pex14Δ mutant expressing NcPEX14 (A). An identical blot was also decorated with anti-ScPEX14 antibodies (B). NcPEX14 was detected by both antibodies (black arrows) whereas yeast PEX14 was only recognized by anti-ScPEX14 antibodies (open arrows). The second band of approximately 29 kDa represents a stable degradation product of ScPEX14. Analysis of post-nuclear supernatants from N. crassa wild type and a pex14Δ mutant also revealed a PEX14-specific band of 47 kDa (A). (C) PEX13 antibody test. Antisera against a recombinant GST fusion of the SH3 domain of PEX13 were similarly tested for specificity using whole-cell extracts from two-hybrid strain PJ69-4a that had expressed Gal4 DNA-binding or activation domain fusions of the SH3 domain of PEX13 (AD-SH3, BD-SH3), full-length PEX13 (BD-PEX13), or PEX14 (AD-PEX14). N.c. = N. crassa extract; S.c. = S. cerevisiae extract.

Figure 3 (jpg)

Figure 4. Expression of GFP-HEX1.
Correct expression of the fusion protein was determined by Western blotting using anti-HEX1 (left panel) and anti-GFP (right panel) antibodies. The amount of endogenous HEX1 exceeded that of GFP-HEX1.

Figure 4 (jpg)