Liposome‐mediated introduction of proteins into protoplasts of the yeast Hansenula polymorpha as a possible tool to study peroxisome biogenesis

Low pH‐induced fusion between liposomes and protoplasts of the yeast Hansenula polymorpha was demonstrated using a fluorescent assay and freeze‐etch techniques. By this method foreign proteins could be introduced into the cytosol of the protoplast. For instance, after fusion of glucose oxidase‐containing liposomes with protoplasts, activity of this enzyme was demonstrated cytochemically in the cytosol of the resulting protoplasts. Similar results were obtained when ferritin‐containing liposomes were used. Incorporation of foreign proteins into liposomes was not a prerequisite for their introduction into the cytosol of protoplasts. In experiments where ferritin was added to protoplast suspensions together with empty liposomes, this protein was also delivered to the protoplast cytosol. However, in the absence of liposomes no uptake of proteins occurred. We tested the potential of this system in our studies on peroxisome biogenesis. Protoplasts of glucose‐grown H. polymorpha remained stable for prolonged periods in osmotically stabilized cultivation media. Peroxisomes in such protoplasts were capable of protein import and assembly of matrix proteins as was demonstrated by the 20‐fold increase in catalase activity after incubation with methanol or ethanol. However, mature alcohol oxidase purified from H. polymorpha introduced into protoplasts of glucose‐grown cells of this organism was not targeted to peroxisomes as demonstrated with (immuno)cytochemical techniques. The enzyme remained present in the cytosol while its activity gradually decreased. Therefore mature alcohol oxidase probably does not expose the right topogenic signal(s) for recognition by its target organelle.