The yeast cell wall provides a first barrier to the environment, confers shape and stability to the cells, and serves as a model for fungal cell wall biosynthesis and function in general. During normal growth, during mating and upon cell surface stress, new wall synthesis is induced by a conserved signaling cascade, the cell wall integrity (CWI) pathway. A signal is initiated by plasma membrane-spanning sensors and transduced through a mitogen-activated protein kinase (MAPK) cascade, which ultimately activates a transcriptional activator, Rlm1. The first part of this thesis analyses the role of this MADS-box transcription factor in the milk yeast Kluyveromyces lactis, which has not been investigated, until now. With respect to the distribution of the upstream CWI sensors, evidence for the existence of a special plasma membrane microcompartment, generally referred to as eisosomes, in the milk yeast is provided in the second part of the thesis.
Regarding the transcription factor KlRlm1, its impact on the physiology of K. lactis seems to be different from its homolog in Saccharomyces cerevisiae, ScRlm1, although it clearly acts in CWI signaling, too. Thus, in contrast to the Scrlm1 mutant, a Klrlm1 deletion is sensitive, rather than hyper-resistant, towards Congo red and Calcofluor white, typical stress agents used in cell wall research. Data on cross-complementation of the two genes in the respective heterologous yeast indicate that KlRlm1 and ScRlm1 each perform their optimal function only in the native host.To investigate the impact of a Klrlm1 deletion on the transcriptional profile of K. lactis, data from total mRNA sequencing were analyzed in comparison to a wild-type strain. Many of the genes identified did not correspond to known Rlm1 target genes in S. cerevisiae, but many relate to other stress responses (e.g. KlGRE1, KlFMP16, KLLA0C05324g, KLLA0F18766g, KlUGX2) and to chitin synthesis (KlCHS1, KlSKT5 and KlYEA1), both probably connected to cell wall composition. The functions of a large group of KlRlm1 dependent genes identified here are yet uncharacterized or lack homologs in S. cerevisiae. The plasma membrane of fungi is a specialized organelle, which is ordered into several lateral domains, which we define as microcompartments, since each is composed of a special combination of proteins in their lipid environment. Such microcompartments are believed to control a variety of signaling (and transport) processes in all sorts of eukaryotic cells. Microcompartmentalization is also observed in the yeast plasma membrane, e.g. displayed by the CWI sensors in K. lactis, as shown in this thesis. Since distribution of the latter sensors is reminiscent of that of eisosomes, it was also investigated by live-cell fluorescence microscopy, how KlPil1, KlLsp1 and KlSur7 (all homologs of eisosomal proteins in S. cerevisiae) are distributed. Since they form the typical membrane patches, which are not present in deletion mutants of KlPIL1, the major structural component of eisosomes, one can conclude, that eisosomal microcompartments form in K. lactis and are composed similar to their counterparts in S. cerevisiae. The CWI sensors are excluded from these structures and form their separate microcompartments. The exact physiological function of eisosomes in fungi is still a matter of debate and future studies in K. lactis may help to address this role.
| Identifer | oai:union.ndltd.org:uni-osnabrueck.de/oai:repositorium.ub.uni-osnabrueck.de:urn:nbn:de:gbv:700-2014092412830 |
| Date | 24 September 2014 |
| Creators | Meyer, Sascha |
| Contributors | Prof. Dr. Jürgen Heinisch, apl. Prof. Dr. Hans Merzendorfer |
| Source Sets | Universität Osnabrück |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis |
| Format | application/zip, application/pdf |
| Rights | http://rightsstatements.org/vocab/InC/1.0/ |
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