Difference between revisions of "Part:BBa K2271060"

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Snc1

Usage and Biology

Figure 1. A diagram of the general domain structure of Snc1. V is a variable domain which is not important for the binding to the t-SNARE. TM is the transmembrane domain. H1 and H2 are the α-helical segments, forming the SNAREpin with the t-SNARE Gerst et al. (1997)

This part is a truncated version of the v-SNARE (vesicle- synaptosome-associated-Soluble N-ethylmaleimide-sensitive-factor Attachment REceptorprotein) Snc1. Snc1 is in the wildtype form involved in the fusion of Golgi-derived secretory vesicles with the plasma membrane. Domains of the protein are a variable domain which is not important for the binding to the t-SNARE, H1 and H2 are the a-helical segments (forming the SNAREpin with the t-SNARE) and the transmembrane domain (Gerst Paper). For our approaches we used a truncated version without the transmembrane domain. This Snc1 truncation was fused to the N-Terminus of different peroxisomal membrane anchor (Pex15 /Pex26) to secrete the compounds of this compartment (Bild).

Experimental design

For testing this part we used a fusion with the N-Terminus of a peroxisomal membrane anchor. We co-expressed this construct with GUS-PTS1 to perform a GUS Assay.(oder: We performed a GUS-Assay by targetting GUS(beta-Glucuronidase) to the peroxisome using a GUS-PTS1.) The secreted GUS in the supernatant was measured with the turnover of 4-methylumbelliferyl-beta-D-glucuronide to 4-methyl umbelliferone (4-MU). The fluorescent 4-MU was measured with a plate reader (excitation: 365 nm, emission: 465 nm).

Results

Different peroxisomal membrane anchors were tested using the GUS-Assay. The highest activity of GUS could be measured in the supernatant of Pex15 (Link to part) as a Membrane Anchor.

Figure 3. Relative fluorescence units per minute (RFU/min) measured for supernatants of different S. cerevisiae strains. The fluorescence was measured for 12 hours in intervals of 10 minutes with an excitation of 365 nm and an emission of 465 nm. For the strain BY4247 (wt) which was used as the background strain the fluorescence did not increase over measured period. The lysis controls (GUS-PTS1; ∆Pex11 GUS-PTS1) showing a lower activity than the samples of strain with peroxisomes decorated with Snc1. The highest activity could be measured in the strain using Pex15 with a linker as a membrane anchor (Pex15 L). The assay was performed in three technical replicates.

Sequence and Features