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## Comparison of β1- and β2-subunits of Rab geranylgeranyl transferase from Arabidopsis thaliana

### Marta Wnuk

#### Abstract

Rab geranylgeranyltransferase (Rab GGTase) is an enzyme responsible exclusively for double geranylgeranylation of Rab proteins. Rab proteins are small GTPases that regulate many different steps of vesicular transport. Prenylation allows these proteins to associate with lipid bilayer of transport vesicles, therefore it is necessary for their proper functioning. The Rab GGTase complex is build of α and β subunits forming a catalytic core and the third component - protein substrate presenting REP (Rab Escort Protein). Arabidopsis thaliana (plant model organism) has two genes coding for each of the core subunits and one gene coding for Rab Escort Protein. The aim of this study was to compare the β1 and β2 subunits of Rab GGTase from Arabidopsis thaliana. Experiments were performed using wild-type line (Columbia-0 ecotype) and mutant lines with T-DNA insertions in genes encoding both β subunits of Rab GGTase: SALK_015871 (rgtb1-1), SALK_125416 (rgtb1-2), SALK_027208 (rgtb2-1), SALK_149200 (rgtb2-2). The theoretical introduction of this thesis contains the review of the available data on Rab GGTase and the two other protein prenyltransferases: GGTase-I and FTase. This chapter, includes also a short description of other posttranslational lipid modifications of proteins, the process of vesicular transport, Rab family of small GTPases and their functions in plant cells. In the next chapter the results obtained in my experimental work are presented. The comparison of the nucleotide and amino acid sequences of both β subunits of Rab GGTase revealed high homology of these proteins. All mutant lines used in research have been characterized. The polymerase chain reactions (PCRs) confirmed the presence of T-DNA insertions in each mutant line. Sequencing of PCR products allowed to determine the 2 localization of T-DNA insertions in the two rgtb2 mutant lines. In both cases the insertion was found within the relevant gene, in the sixth (rgtb2-1) and ninth exon (rgtb2-2) respectively. Morphological observations of mutant lines have also been performed. Both rgtb1 mutant lines exhibited an identical characteristic phenotype changes and both rgtb2 mutant lines did not differ significantly from wild-type plants. Contrary to previous reports, our results show that homozygous rgtb1-1 and rgtb1-2 plants can produce siliques with a small number of seeds. Expression profiles for AtRGTB1 and AtRGTB2 genes was obtained using semi-quantitative RT-PCR. In both rgtb1 mutant lines expression of AtRGTB1 gene was not detected, whereas the levels of AtRGTB1 mRNA were comparable in different plant organs of wild-type line and rgtb2 mutants. Surprisingly, the result of this experiment indicate the presence of AtRGTB2 mRNA in different organs of rgtb2-2 mutants. This may mean that rgtb2-2 mutant produce a certain amount of functional β2 subunit. In a second rgtb2 mutant line (rgtb2-1) expression of AtRGTB2 gene was not detected. In PCR reaction with primers complementary to the gene encoding the β2 subunit the second PCR product was detected. It may be the product of alternative splicing, but this observation should be confirmed by sequencing. Results of this experiment also revealed the higher accumulation of AtRGTB1 mRNA than AtRGTB2 mRNA in all examined organs of wild-type plants. Western blot analysis using rabbit primary antibodies anti-RABGGTβ was performed to confirm the presence of the β2 subunit protein in the rgtb2-2 mutants. Unfortunately, this experiment has not given satisfactory results. Morphological observations of root hairs in all rgtb1 and rgtb2 mutants have been performed. Various structure anomalies have been observed. Some of them were present in the roots of wild-type plants, but with lower frequency. Results of statistical analysis lead to the conclusion that in all mutant lines total number of root hairs exhibiting structure anomalies is substantially higher than in wild-type plants. Because of this evident phenotype we examined the process of endocytosis in root hairs of rgtb1 and rgtb2 mutants and wild-type plants using the fluorescent styryl dye FM1_43. The dye accumulated much faster inside the root hairs of rgtb1 and rgtb2 mutants than in root hairs of wild-type plants. The results of both experiments show that reducing the activity of Rab GGTase, caused by mutations in genes encoding both β subunits of this enzyme, disrupts the processes associated with vesicular transport in roots. An important aim of the research was to construct the double mutant carrying T-DNA insertions in both genes encoding β subunits of Rab GGTase. In the F2 generation of two 3 independent crosses of rgtb1 plants with rgtb2 plants the double mutant was not detected. The statistical analysis of the results confirms that double mutation is lethal. The next experiments were performed to determine the stage on which double homozygotes are eliminated from the population, but they did not give clear answer to this question. The thesis contains also the description of materials and methods used in the experimental work and a brief summary of the results.
Record ID
WUT9b8a9d6d47d4468e833dc3336ce04866
Diploma type
Master of Science
Author
Marta Wnuk Marta Wnuk,, Undefined Affiliation
Title in Polish
Podjednostki β1 i β2 kompleksu Rab Geranylogeranylotransferazy u Arabidopsis thaliana - charakterystyka porównawcza
Supervisor
Maria Bretner (FC/CDSB) Maria Bretner,, Chair of Drug and Cosmetics Biotechnology (FC/CDSB)Faculty of Chemistry (FC)
Certifying unit
Faculty of Chemistry (FC)
Affiliation unit
Department Of Drug Technology And Biotechnology (FC/CDSB)
Study subject / specialization
, Biotechnologia Chemiczna - Leki i Kosmetyki
Language
(pl) Polish
Status
Finished
Defense Date
25-06-2012
Issue date (year)
2012
Keywords in Polish
prenylacja, Rab geranylogeranylotransferaza (Rab GGTaza), białka Rab, podjednostka β Rab GGTazy, transport pęcherzykowy, mutanty insercyjne.
Keywords in English
prenylation, Rab geranylgeranyltransferase (Rab GGTase), Rab proteins, Rab GGTase β subunit, vesicular transport, insertional mutants.
Abstract in Polish
urn:pw-repo:WUT9b8a9d6d47d4468e833dc3336ce04866