Appendix 3 Review of Selected Papers and Discussion of the Research And

Appendix 3

Review of selected papers and discussion of the research and other achievements

Liliana Surmacz, PhD

Institute of Biochemistry and Biophysics Polish Academy of Sciences Department of Lipid Biochemistry Pawińskiego 5a 02-106 Warsaw, Poland

Warsaw 2017 Appendix 3 L. Surmacz

1. Name and surname: Liliana Surmacz

2. Diplomas, scientific degrees held - including the name, place and year of acquisition and the title of the doctoral dissertation:

2004 Ph.D. in Biology - Molecular Biology, Nencki Institute of Experimental Biology PAS in Warsaw, The title of Ph.D. thesis „Rab7 in Paramecium cells: studies on gene, protein and localization during endocytosis”, Supervisor: Prof. Elżbieta Wyroba

1995 M.Sc. in Biology - Microbiology, University of Lodz, Faculty of Biology and Earth Sciences, The title of M.Sc. thesis: „The use of an enzyme immunoassay APAAP for the identification of markers that differentiate human lymphocytes”, Supervisor: Prof. Wiesława Rudnicka

3. Information on employment in scientific institutions: science 10. 2006 till present Assistant Professor at the Department of Lipid Biochemistry, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland. 09.2008 – 05.2010 maternity leave 09.2006 – 10.2006 biologist at the Department of Lipid Biochemistry, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland. 12.1995 – 08.2006 assistant at the Laboratory of Cell Membrane Physiology, the Nencki Institute of Experimental Biology PAS, Warsaw, Poland 01.1998 – 01.2000 maternity leave

4. Description of the achievement under the Article 16.2 of the Act of 14 March 2003 on academic degrees and academic title and degrees and title in art (Journal of Laws, item 882, 2016 and item 1311, 2016): a) title of the scientific achievement, The scientific achievement consists of five papers published in the journals listed by the Journal Citation Report, total IF matching the year of publication is: 26.581 (WoS); total points (according to the list of the Ministry of Science and Higher Education, MSHE): 180; times cited: 58 (WoS).

Polyisoprenoids as eukaryotic "superlipids" - characteristic of mechanisms of biosynthesis and the search for cellular functions. b) authors, title of publication, year of publication, name of publisher:

1. Surmacz L*, Swiezewska E. (2011) Polyisoprenoids - Secondary metabolites or physiologically important superlipids? Biochem Biophys Res Commun. 407, 627-632.

IF2011: 2.484 (WoS); 20 points (MSHE); times cited: 37 (WoS)

Appendix 3 L. Surmacz

2. Surmacz L*, Plochocka D, Kania M, Danikiewicz W, Swiezewska E. (2014) cis-Prenyltransferase AtCPT6 produces a family of very short-chain polyisoprenoids in planta. Biochim Biophys Acta. 1841, 240-250.

IF2014: 5.162; (WoS); 35 points (MSHE); times cited: 10 (WoS)

3. Akhtar TA*, Surowiecki P, Siekierska H, Kania M, Van Gelder K, Rea KA, Virta LKA, Vatta M, Gawarecka K, Wojcik J, Danikiewicz W, Buszewicz D, Swiezewska E, Surmacz L* (2017) Polyprenols are synthesized by a plastidial cis-prenyltransferase and influence photosynthetic performance. Plant Cell, 29, 1709-1725.

IF2016: 8.688 (WoS); 45 points (MSHE); times cited: 1 (WoS)

4. Brasher MI, Surmacz L, Leong B, Pitcher J, Swiezewska E, Pichersky E, Akhtar TA.(2015) A two-component enzyme complex is required for dolichol biosynthesis in tomato. Plant J. 82, 903- 914.

IF2015: 5.468 (WoS); 45 points (MSHE); times cited: 9 (WoS)

5. Surmacz L*, Wojcik J, Kania M, Bentinger M, Danikiewicz W, Dallner G, Surowiecki P, Cmoch P, Swiezewska E. (2015) Short-chain polyisoprenoids in the yeast Saccharomyces cerevisiae - New companions of the old guys. Biochim Biophys Acta 1851, 1296-1303.

IF2015: 4.779 (WoS); 35 points (MSHE); times cited: 1 (WoS)

*corresponding author c) presentation of the scientific objectives of the publications listed above, the results obtained, and possible applications

The scientific achievement is a collection of five thematically closely related papers presenting the results of my studies performed at the Department of Lipid Biochemistry, Institute of Biochemistry and Biophysics Polish Academy of Sciences. The main objective of the studies summarized herein was the identification, molecular characterization and elucidation of the cellular role of enzymes involved in polyisoprenoid biosynthesis. These studies were motivated by the vital role polyisoprenoid lipids play in the cells (among others as obligate cofactors of protein glycosylation) and concomitantly by the extremely limited knowledge on their biosynthetic mechanisms, especially in plant cells. When performing this project I cooperated with research groups from Poland and abroad, i.e. the group of Prof. Witold Danikiewicz from the Institute of Organic Chemistry of the Polish Academy of Sciences in Warsaw and the group of Dr. Tariq Akhtar from the University of Guelph, Canada. Studies described in the papers constituting the presented scientific achievement were financially supported by the projects submitted by me and performed under my leadership: POL-POSTDOC II grant "The role of protein prenylation and prenylated lipids in the vesicular transport in plants” provided by the Polish 3

Appendix 3 L. Surmacz

Ministry of Science and Higher Education (2006 – 2011) and National Centre of Science OPUS 2 grant "Implications of gene multiplicity of Arabidopsis cis-prenyltransferases on the polyisoprenoid alcohols biosynthesis and plant stress tolerance" (2012 – 2016).

Polyisoprenoid alcohols are biopolymers present in the cells of all living organisms. Despite the fact that polyisoprenoids have been studied extensively for more than 50 years, our knowledge of their properties, the mechanisms of biosynthesis and their role in the cell is still far from being complete. Polyisoprenoids have important biological functions during protein post- and co-translational modifications, namely they serve as obligate cofactors in the biosynthesis of glycosyl-phosphoinositol (GPI) anchor and protein N-, O- and C-glycosylation [Burda et al., 1999; Pattison et al., 2009; Nothaft at al., 2010] as well as donors of isoprenoid groups in protein prenylation [Swiezewska et al., 1993]. Moreover, they are implicated in cell adaptation to adverse environmental conditions [Bajda et al., 2009] possibly via modulation of the properties of biological membranes by increasing their permeability and fluidity and enhancing membrane fusion [summarized in Swiezewska and Danikiewicz, 2005]. Importantly, mutations in genes encoding enzymes of the polyisoprenoid biosynthesis pathway lead to severe metabolic disorders in humans (CDG, Congenital Disorders of Glycosylation type I) [summarized in Buczkowska et al., 2015] while some of them are lethal in plants [summarized in Hemmerlin et al., 2012]. Polyisoprenoid alcohols found in cells vary in their chain length - depending on the origin polyisoprenoid chains consist of from 5 to more than 100 isoprene residues, with a hydroxyl group at one end and hydrogen at the other (α- and ω-isoprene residue, respectively) [Swiezewska and Danikiewicz, 2005]. Depending on the presence of the double bond in the α-isoprene residue, these compounds are subdivided into polyprenols (α-unsaturated, Pren) and dolichols (α-saturated, Dol). Polyprenols occur mainly in bacteria cells and plant photosynthetic tissues whereas dolichols - in animal and yeast cells and in plant roots. It is worth underlining that in eukaryotic cells dolichols and polyprenols are always found as mixtures of homologues (named ‘families’), moreover, dolichols are accompanied by traces of polyprenols of the same chain-length. Polyisoprenoids are accumulated in the form of free alcohols and/or esters of carboxylic acids along with a small fraction of mono- and diphosphates. The content of polyisoprenoids increases during the life span of organisms and upon pathological conditions or environmental stress.

Based on the configuration of the double bonds polyisoprenoid alcohols are classified into three main groups: 1) di-trans-poly-cis – bactoprenol (in most bacteria composed of 11 isoprene units) and plant polyprenols containing 5-9 isoprene units (i.u.) or more than 16 i.u. and animal, yeast and plant dolichols 2) tri-trans-poly-cis – polyprenols containing 9-15 i.u.

Appendix 3 L. Surmacz

3) all-trans – solanesol (all-trans Pren-9) isolated from Solanaceae plants [Hemming, 1983] and all- trans Pren-7 from Saccharomyces cerevisiae [Surmacz et al, 2015], oligoprenols: geraniol (2 i.u.), farnesol (3 i.u.) and geranylgeraniol (4 i.u.) also belong to this group.

Biosynthesis of polyisoprenoids occurs in three steps: 1) Formation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) – five- carbon precursors of isoprenoids; 2) Elongation of polyisoprenoid chain by subsequent condensation of IPP molecules to obtain polyprenyl diphosphate/polyprenol; 3) Reduction of the double bond in the α residue of polyprenol to form dolichol.

Step 1. All isoprenoids are formed from the common precursor, isopentenyl diphosphate (IPP), which in plants is synthesized via two pathways operating in parallel: the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathway. Both IPP-generating pathways are compartmentalized in plant cells - the enzymes of the MVA pathway are present in the cytoplasm whereas those of the MEP pathway – in plastids [summarized in Hemmerlin et al., 2012]. In contrast to plant cells, in archaebacteria [Smit and Mushegian, 2000], some gram-positive bacteria [Wilding et al., 2000], yeasts [Denbow et al., 1996] and animals [Kovacs et al., 2002] IPP is derived exclusively from the MVA pathway, whereas in most gram-negative bacteria (e.g., Bacillus subtilis and Escherichia coli) [Rohmer et al., 1993], cyanobacteria [Proteau, 1998], and green algae [Disch et al., 1998] – from the MEP pathway.

Step 2. Synthesis of the polyisoprenoid backbone occurs by the sequential head-to-tail condensations of an IPP molecule to its isomer dimethyl allyl diphosphate (DMAPP) and is catalyzed sequentially by two types of enzymes known as prenyltransferases. Initially the all-trans precursor - geranyl (C10), farnesyl (C15) or geranylgeranyl diphosphate (C20) - is formed by a specific trans-prenyltransferase (TPT), and subsequently it is elongated by a dedicated cis-prenyltransferase by successive additions of IPP molecules [Swiezewska and Danikiewicz, 2005; Liang, 2009]. These catalytic processes occur in association with the membrane, either of the bacterial cell or the endoplasmic reticulum of the eukaryotic cell [Kharel and Koyama, 2003]. So far, only a few prenyltransferases have been crystallized, namely three eukaryotic trans-prenyltransferases from Arabidopsis (AtGGPPS11, AtGFPPS2, and AtPPPS2) [Wang et al., 2016] and several bacterial cis-prenyltransferases: undecaprenyl diphosphate synthase (UPPS) of Micrococcus luteus [Fujihashi et al., 2001], Escherichia coli [Ko et al., 2001], decaprenyl diphosphate synthase (DPPS) and cFPPS of Mycobacterium tuberculosis [Wang et al., 2008]. Interestingly, prokaryotic cis-prenyltransferases synthesize only one product in contrast to all so far characterized eukaryotic counterparts which produce mixtures (‘family’) of homologues. The first identified cis-prenyltransferase was undecaprenylPP synthase (UPPS) from Micrococcus luteus B-P 26 5

Appendix 3 L. Surmacz

[Shimizu et al., 1998]. Bacteria only have a single gene encoding CPT involved in biosynthesis of a single polyprenol, in most bacteria it is Pren-11 (undecaprenol, bactoprenol) [Rush et al., 2010] with the exception of Halobacterium halobium or Mycobacterium smegmatis, where single Dol-12 or Pren-10 were found, respectively [Lechner et al, 1985; Wolucka et al, 1994]. Yeast Saccharomyces cerevisiae have two active cis-prenyltransferases (Rer2 and Srt1) with different intracellular localizations (endoplasmatic reticulum and lipid bodies) and different product specificity – family of dolichols with dominating Dol-15 (Rer2) and Dol-21 (Srt1) [Sato et al., 1999; 2001]. Single CPT was identified in humans (hCIT/HDS) [Endo et al., 2003; Shidras et al., 2003] and the parasite Giardia lamblia (Gl-CPT) [Grabinska et al., 2010]. HDS catalyzes formation of one Dol family consisting of six dolichols with Dol- 19 dominating while Gl-CPT is responsible for synthesis of a ‘narrow’ family of dolichols (Dol-11 and Dol- 12). Seven genes encoding CPT were identified in tomato (SlCPT1 – SlCPT7). Only one of them, SlCPT3, is involved in biosynthesis of the dolichol family with dominating Dol-16. The remaining SlCPTs produce short-chain isoprenoids [Akhtar et al., 2013]. Two CPTs from Hevea brasiliensis (HRT1 and HRT2) [Asawatreratanakul et al., 2003], and three from Taraxacum koksaghyz (TkCPT1– TkCPT3) [Schmidt et al., 2010] are responsible for formation of long-chain polyisoprenoids of natural rubber. Only one CPT (LAA66) has been described in Lilium logiflorum [Liu et al., 2011] which in fact does not preclude the existence of a family of CPT encoding genes in this plant. Nine genes encoding putative CPTs (AtCPT1 – AtCPT9) have been predicted in the Arabidopsis genome, moreover, possible redundancy of their cellular function has been proposed. Until 2006 when I joined the project on Arabidopsis CPT only one AtCPT had been partially characterized at the molecular level - recombinant AtCPT1 (called DPS or ACPT) was shown to be involved in the synthesis of a single Dol-18 [Cunillera et al., 2000] or Pren-24 [Oh et al., 2000] in vitro. Our recent in vivo studies have shown that in the heterologous yeast system, AtCPT1 synthesizes a dolichol family with Dol-18 dominating, while in the heterologous plant system - Nicotiana benthamiana – a dolichol family with Dol-21 dominating [Surmacz and Surowiecki, unpublished data ]. Oh et al. [2000] observed expression of AtCPT1 in all analyzed plant tissues whereas Cunillera et al. [2000] found it only in roots. Our studies confirmed that AtCPT1 is expressed only in the root tissue [Surmacz and Swiezewska, 2011]. The results of our studies on the selected AtCPTs are presented further in this summary. Step 3. The final product of cis-prenyltransferase, polyprenyl diphosphate, is dephosphorylated and subsequently its α-isoprene residue is reduced by polyprenyl reductase - PPRD in plants [Jozwiak et al., 2015] and SRD5A3 in mammals [Cantagrel et al., 2010] and yields dolichol, which is further converted to Dol-P by dolichol kinase [Rossignol et al., 1983].

Numerous aspects of polyisoprenoid biosynthesis discussed above have been described in our review Surmacz L, Swiezewska E. (2011) Polyisoprenoids – Secondary metabolites or physiologically important superlipids? Biochem Biophys Res Commun. 407, 627-632. On the one

Appendix 3 L. Surmacz hand this paper presents a brief summary of the up-to-date knowledge available when I decided to enter the field of polyisoprenoids and on the other hand it constitutes an outline clearly showing the gaps to be filled and indicates the future directions to be followed. This review paper also contains a simple experimental section with preliminary characterization of putative Arabidopsis cis-prenyltransphase encoding genes which indicated (RT-PCR analysis) that AtCPT1 – AtCPT9 show tissue-specific expression. Isoforms AtCPT3 and AtCPT7 are expressed in all the analyzed tissues (root, leaf, stem, flower) at a high level, while AtCPT4, AtCPT5 and AtCPT8 are expressed only in flowers and AtCPT1, AtCPT6 and AtCPT9 only in roots. AtCPT2 is expressed in leaves, flowers and roots at a high level, while it shows a very low level of expression in the stem. AtCPTs display a high level of identity of amino acid sequence (~29 – 80%) to each other and high sequence homology to both yeast CTPs, 49 – 55% to Rer2 and 37 – 50% to Srt1, respectively. Interestingly, this analysis indicated that AtCPTs exhibit a low similarity to the LEW1 (leaf wilting 1) protein described in 2008 as the tenth Arabidopsis cis- prenyltransferase [Zhang et al., 2008]. Interesting aspects of the function (other than originally believed) of the LEW1 protein and its homologues from other organisms will be discussed further in this summary. It should be underlined that although the presence of CPT families in plants, e.g. Arabidopsis [Lange and Ghassemian, 2003], was suggested by the genomic sequence analysis, the identification of the function of individual homologues has not been completed and the question of their functional redundancy remains open. In addition, a broad set of data describing the polyisoprenoid profile of numerous plant species has been collected during past decades at the Department of Lipid Biochemistry IBB PAS. Multifamily spectra noted for various plant genera suggested the involvement of several rather than one CPT in their formation. Taken together, the lack of molecular characterization of plant CPTs and our earlier biochemical observations together with a unique set of polyisoprenoid standards available at the Department prompted me to undertake a new line of molecular research focused on polyisoprenoids. Shortly after publishing this review, I received an invitation from Dr. Tariq Akhtar, University of Guelph, Canada, to join a scientific collaboration. Since his scientific plans seemed interesting and close to my own research interests we decided to establish the cooperation. The joint project was aimed at characterizing proteins involved in polyisoprenoid biosynthesis in plants. These efforts, possibly due to the complementary research expertise and two plant models (tomato and Arabidopsis) used by our groups, resulted in the publication of two papers with shared authorship [Brasher et al., 2015 and Akhtar et al., 2017]. These papers, included as a part of the presented scientific achievement, are discussed below. Scientific cooperation with Dr. Akhtar is being continued. The divergent profile of polyisoprenoids in plant tissues, i.e. the occurrence of dolichols in roots and polyprenols in green tissues of plants, has prompted us to determine which CPT is responsible for their synthesis. Furthermore, the complexity of the mixtures of both polyprenols and dolichols (‘multifamily’ profile due to the presence of several homologs) in plant tissues suggested the involvement of more than one CPT in their synthesis. Our earlier studies revealed that three families of dolichols are present in

Appendix 3 L. Surmacz

Arabidopsis roots: short- (Dol-13 dominating), medium- (Dol-16 dominating) and long-chain (Dol-21 or Dol-23 dominating) [Jozwiak et al., 2013]. This profile seems even more complex since a small amount of Dol-7, a representative fourth family of very short-chain dolichols has also been detected by us [Surmacz et al., 2014]. In contrast to roots, the presence of only one family of polyprenols, with Pren-10 dominating, was observed by us in Arabidopsis leaves [Akthar et al., 2017]. Based on the tissue-specific expression of AtCPT and the phylogenetic analysis of CPT from various organisms we have decided to focus on two Arabidopsis cis-prenyltransferases: AtCPT6, expressed only in roots and phylogenetically closely related to yeast CPT involved in dolichol biosynthesis, and AtCPT7, expressed solely in photosynthetic tissue and phylogenetically distinct from yeast CPTs but similar to bacterial CPT (UPPS). Characterization of both these enzymes was performed at the molecular and biochemical level in 2 systems: heterologous - in yeast cells and homologous - in planta.

AtCPT6 is the first ever plant cis-prenyltransferase characterized in planta in the paper Surmacz L, Plochocka D, Kania M, Danikiewicz W, Swiezewska E. (2014) cis-Prenyltransferase AtCPT6 produces a family of very short-chain polyisoprenoids in planta. Biochim Biophys Acta. 1841, 240-250). Expression of AtCPT6 in the temperature-sensitive yeast mutant rer2Δ, with a deletion of the gene encoding yCPT – Rer2, indicated that AtCPT6 catalyzed the synthesis of polyisoprenoids which are accumulated in the cells as mixture of polyprenols/dolichols composed of 6 to 8 i.u., with Pren-7/Dol- 7 dominating. This product specificity was confirmed in the native system of A. thaliana. The content of Pren-7/ Dol-7 in roots of Arabidopsis T-DNA insertion mutant plants (NASC) with an inactivated AtCPT6 gene (cpt6-1 and cpt6-2) was considerably lower than in wild type plants while AtCPT6 overexpression significantly increased the accumulation of these lipids in roots of the CPT6-OE mutant. The structural model of AtCPT6, obtained by homology modeling, further confirmed the capability of AtCPT6 to synthesize short-chain polyisoprenoid products. Similar activity of AtCPT6 (called AtHEPS, cis,trans- mixed heptaprenyl diphosphate synthase) was demonstrated in vitro [Kera et al., 2012]. Using the specific AtCPT6 anti-peptide antibody designed by us (custom synthesis by Agrisera, Vännäs, Sweden) we have shown that AtCPT6, alike most of the previously described eukaryotic CPTs, including yeast Rer2, is associated with the endoplasmic reticulum. Although AtCPT6 catalyzes the synthesis of very short-chain dolichols both in yeast and plant cells, it only partially complements Rer2 function in the rer2Δ mutant, i.e. it restores yeast growth but does not restore glycosylation of carboxypeptidase Y (CPY, secreted protein with four N-glycosylation sites, used as a marker of yeast protein glycosylation status). The role of AtCPT6 in protein glycosylation in plants has not been clarified yet and requires further studies. Similarly, the physiological role of AtCPT6 is not known, however, its function in plant response to environmental stress has been suggested [Kera et al., 2012].

Characteristics of AtCPT7 have been described in the very recent paper Akhtar TA, Surowiecki P, Siekierska H, Kania M, van Gelder K, Rea KA, Virta LKA, Vatta M, Gawarecka K, Wojcik J,

Appendix 3 L. Surmacz

Danikiewicz W, Buszewicz D, Swiezewska E, Surmacz L (2017) Polyprenols are synthesized by a plastidial cis-prenyltransferase and influence photosynthetic performance. Plant Cell, 29, 1709- 1725. We have proved that AtCPT7 is responsible for the synthesis of a family of polyprenols (Pren-9 – Pren-11, Pren-10 dominating) accumulated in Arabidopsis leaves – they were absent in homozygous cpt7-/- mutant plants and their content was considerably decreased in AtCPT7 RNAi silenced lines while the level of these lipids was significantly increased in mutants overexpressing AtCPT7 (CPT7-OE). Elucidation of AtCPT7 function using the functional complementation assay of the rer2Δ yeast strain appeared more difficult than expected. Expression of the full AtCPT7 sequence did not restore yeast growth at higher temperature. Comparison of amino acid sequences of CPT from various organisms (bacteria, plants and animals) revealed the presence of a chloroplast targeting sequence in AtCPT7. Interestingly, five out of the seven tested truncated AtCPT7 forms, devoid of 30 to 67 N-terminal amino acids overlapping with a potential chloroplast targeting sequence, upon expression in the rer2Δ strain were able to synthesize polyisoprenoids (mixture of prenols and dolichols) composed of 9 -12 isoprene residues, which led to restoration of both growth of rer2Δ at elevated temperature and normal CPY glycosylation. Such an effect was not observed for the expression of the shortest truncated AtCPT7 variants devoid of 76 N-terminal aa. In vitro enzyme activity assays confirmed the role of AtCPT7 in synthesis of lipids composed of 10-11 i.r. and showed that FPP and/or GGPP might serve as precursors of this reaction. 1H NMR structural analysis of Pren-10 isolated from Arabidopsis leaves revealed the tri- trans-poly-cis configuration and clearly indicated that GGPP, synthesized mostly in plastidial compartment of plant cells, is a precursor for AtCPT7 in planta. This observation is consistent with the intracellular localization of AtCPT7, which was detected in the stroma of chloroplasts, while its lipid products were mainly accumulated in the thylakoid membranes. The absence or deficiency of polyprenols synthesized by AtCPT7 in the cpt7 insertion or RNAi mutants appears to alter thylakoid membrane dynamics and reduces plant photosynthetic capacity most probably due to the defective linear photosynthetic electron transport of the mobile plastoquinone electron acceptor. This novel observation linking polyprenols with photosynthesis seems very interesting. In contrast to dolichols, whose role in protein glycosylation is well known, cellular function of polyprenols has remained elusive. The results of our study shed new light on the role of these "secondary metabolites" in plant physiology, and might be considered in future strategies aimed at increasing plant productivity. Interestingly, our recent observations might explain previously suggested role of polyprenols in plant reproduction and environmental fitness [Hallahan DL, Coldren Ch, Flint D, Wang H - patents No CA2381020A1, EP1214338A2, US6645747, US7273737, US20040152158, US20080096276, WO2001021650A2, WO2001021650A3].

Taken together, our results revealed that AtCPT7 is responsible for biosynthesis of polyprenols in leaves, whereas AtCPT6 – for very short polyprenols and dolichols in roots. Moreover, neither of these enzymes is directly involved in the biosynthesis of dolichols predominant in roots (dolichol families with Dol-16 and Dol-21 dominating, respectively). Our so far unpublished studies revealed that in the 9

Appendix 3 L. Surmacz heterologous system the remaining root AtCPTs, AtCPT1 and AtCPT9, catalyze the synthesis of two dolichol families with Dol-18 and Dol-13 dominating, respectively, and their expression in rer2Δ mutant restores both yeast growth and protein glycosylation [Surmacz and Surowiecki, in preparation]. The profile of polyisoprenoid lipids extracted from yeast transformed with respective genes encoding root AtCPTs (AtCPT1, -6 and -9) only partially matches that observed in planta in A. thaliana roots. This may suggest that polyisoprenoid biosynthesis in plants is more complex and that yet uncharacterized plant regulators other than CPT are necessary to modulate elongation of polyisoprenoid chains.

A good example of such long sought regulators of cis-prenyltransferase activity and protein glycosylation is Nogo-B receptor (NgBR) recently identified in mammalian cells [Harrison et al., 2011; Park et al., 2014]. It does not possess CPT activity per se but instead serving the function of an accessory protein it interacts with the hCIT (human CPT), enhances hCIT protein stability and promotes Dol-P production [Harrison et al., 2011]. Moreover, the yeast NgBR homologue – NUS1 in a similar manner regulates yeast CPTs (Rer2 and Srt1) and determines polyisoprenoid chain length [Park et al., 2014 ]. Until recently plant homologs of NgBR/NUS1 were still awaiting characterization. One might speculate that LEW1 protein, previously described as CPT [Zhang et al., 2008] might fulfill this function and, as already mentioned, instead of a low sequence similarity to the AtCPT family [Surmacz and Swiezewska, 2011] it displays high similarity to human Nogo-B receptor (63%) and yeast NUS1 (57%). Initially it was postulated that LEW1 is involved in dolichol biosynthesis, protein glycosylation and plant stress response although LEW1 only partially complemented the rer2Δ mutant, and its CPT activity is negligible [Zhang et al, 2008]. Our so far unpublished studies revealed that LEW1 does not restore growth or protein glycosylation in rer2Δ yeast strain and does not possesses CPT activity, while on the other hand, upon concomitant expression with selected AtCPT it promotes the synthesis of dolichol families with Dol-16 (AtCPT3) or Dol-13 (AtCPT4, -5) dominating, respectively, and restores CPY glycosylation. Individual expression of each of these proteins, either AtCPTs or LEW1 itself, does not complement the absence of Rer2 in rer2Δ mutant. All these results confirm the role of LEW1 as a regulator of CPT activity in Arabidopsis. Studies on LEW1 are carried out under my supervision by Przemysław Surowiecki within the frame of the NSC project (PRELUDIUM 8, 2015 – 2018) "LEW1 as a protein partner for cis-prenyltransferases in polyisoprenoid biosynthesis in Arabidopsis". Studies on identification of plant regulatory proteins interacting with CPTs are also extensively conducted by other research groups. Very recently published results [Kwon et al., 2016] partially confirm interaction of LEW1 and AtCPT3 proteins (At2g17570, called AtCPT1 by the authors). Simultaneous expression of both plant proteins in the double yeast mutant rer2Δsrt1Δ, devoid of both Rer2 and Srt1 activity, effectively complemented the growth of the mutant strain. In addition, microsomes isolated from the double transformants exhibited cis-prenyltransferase activity in vitro.

Appendix 3 L. Surmacz

Our search for plant homologues of NgBR and NUS1 proteins was conducted in parallel in cooperation with Dr Akhtar using a tomato model. The results were described in the joint paper included in my achievement: Brasher MI, Surmacz L, Leong B, Pitcher J, Swiezewska E, Pichersky E, Akhtar TA. (2015) A two-component enzyme complex is required for dolichol biosynthesis in tomato. Plant J. 82, 903-914. Analysis of the tomato genome allowed to identify a gene encoding a protein with 47% identity to LEW1 and 21% identity to NgBR, further designated as SlCPTBP (CPT-Binding Protein). Using yeast two-hybrid and co-immunoprecipitation assays we have demonstrated that tomato SlCPT3 and SlCPTBP interact in vivo and that both these proteins are required for complementation of the growth, restoration of microsomal CPT enzymatic activity and synthesis of the dolichol family (Dol-14 – Dol-18, Dol-15/16 dominating) of the yeast rer2Δ mutant. Additionally, upon simultaneous expression of both these proteins in E. coli we observed formation of a two-component enzyme complex that synthesizes dolichol in vitro. In planta studies revealed that in RNAi lines with reduced expression of SlCPT3 dolichol (Dol-15 – Dol-17) content was reduced (60% in comparison to wild-type plants) whereas the level of the other polyisoprenoid (solanesol) was unaffected. RNAi lines exhibited a pleiotropic phenotype, which included mottled, wilted leaves and stunted growth. Subcellular localization analysis demonstrated that both SlCPT3 and SlCPTBP localize to the ER, however, a minor fraction of SlCPTBP appears to be localized outside the ER. Western blot analysis of subcellular fractions indicates that SlCPTBP partly localizes to the Golgi membrane system. These observations are in agreement with studies in animals which have demonstrated that NgBR resides in both compartments [Harrison et al., 2009]. This also suggests that plant CPTBPs might serve an additional function besides dolichol synthesis in plants, similarly to animal cells where NgBR is implicated in cholesterol trafficking and homeostasis [Harrison et al., 2009]. The homologues of human NgBR and yeast NUS1 have also been identified in other plants. Studies in lettuce (Lactuca sativa) have shown that CPTL2 (NgBR homologue) protein is a scaffolding protein that tethers CPT3 cis-prenyltransferase on endoplasmic reticulum and is necessary for natural rubber biosynthesis in planta [Qu et al., 2015]. Likewise, a NgBR homologue was identified in ginseng (Panax ginseng Meyer) – PgCPLT2 protein in complex PgCPT1 is involved in dolichol biosynthesis [Nguyen et al., 2017].

Careful inspection of the profile of lipids isolated from various strains analyzed during studies on functional complementation of yeast with plant cis-prenyltransferases led us to identification of short- chain polyisoprenoids in addition to the typically detected mixture of dolichols. This observation deserved careful investigation since such components had previously not been described in the literature despite a large number of studies performed with the aid of the yeast model. These compounds were characterized in the paper: Surmacz L, Wojcik J, Kania M, Bentinger M, Danikiewicz W, Dallner G, Surowiecki P, Cmoch P, Swiezewska E. (2015) Short-chain polyisoprenoids in the yeast Saccharomyces cerevisiae - New companions of the old guys. Biochim. Biophys. Acta 1851,

Appendix 3 L. Surmacz

1296-1303 included in this achievement. Polyprenol (Pren-7) consisting of 7 isoprene residues in the trans configuration (accompanied by traces of Pren-6 and -8) was identified in three studied yeast strains (SS328, BY4741 and L5366). Moreover, in two of these strains (SS328, BY4741) a single, bacterial-like polyprenol (Pren-11) composed of 11 isoprene residues in the trans/cis configuration was detected at the stationary phase of growth. Identity of both these polyprenols was confirmed by HPLC/HR-MS, 1H and 13C NMR and biochemically - by metabolic labeling. Furthermore, we demonstrated that simvastatin (inhibitor of the mevalonate pathway – MVA) inhibited biosynthesis of both polyprenols. Results of metabolic labeling with mevalonate together with the inhibitory effect of simvastatin proved that they are synthesized from the MVA pathway-derived precursors. Additionally, we have shown that trans- hexaprenyltranstransferase classified to trans-prenyltransferases (TPT, with a different structure and regulatory mechanisms than CPT), is involved in the biosynthesis of all-trans Pren-7. This was described as the first enzyme involved in coenzyme Q (UQ-6, ubiquinone-6) biosynthesis in S. cerevisiae [Ashby and Edwards, 1990]. It seems plausible to assume that all-trans Pren-7 is a precursor of UQ-6. In contrast, the role of Pren-11 remains unexplained, and the enzyme responsible for its synthesis is unknown. It neither of the two well-characterized yeast CPTs because Pren-11 is present in the cells of both Rer2 and Srt1-deletion strains. Identification of short-chain Pren-11 in yeast cells is a surprising and interesting discovery. Yeast is a model organism that has been in use in polyisoprenoid studies for half a century. It seems that the results of functional complementation of yeast strains with genes encoding CPT from different organisms may require careful reinterpretation. Investigation on the biosynthesis and biological role of yeast Pren-11 and also on Pren-11 identified by us in mammalian cells [Surmacz, unpublished data] are currently continued within the NCN project OPUS11 “Study on the role and biosynthesis of bacterial-like polyprenols in eukaryotic cells " (2017 – 2020), under my leadership.

Although polyisoprenoids were already identified in the 1960s and are extensively studied, there are still many questions about the mechanisms of their formation and their role in the cell. It seems that functions of plant CPTs, which occur in cells as a family of proteins, do not overlap – CPTs are present in different tissues and divergent cellular compartments, and thus might fulfill distinct, non-redundant functions in the cell. Additionally, the mechanism of polyisoprenoid biosynthesis still has a number of poorly understood aspects such as the activity of CPT regulatory proteins, including recently discovered homologues of NgBR or the role of bacterial-like Pren-11 in yeast and animal cells. In summary, I believe the results of my studies presented here have been instrumental in elucidating the mechanisms of polyisoprenoid lipid biosynthesis in plants. I consider my most important achievements to be as follows: 1. Characterization in planta of the two enzymes synthesizing polyisoprenoids in Arabidopsis – this clearly demonstrates differences in the profile of their products and suggests different roles in the cell. 2. Demonstration that plant CPT (SlCPT in tomato) activity requires formation of a complex with CPT binding protein (NgBR homologue) – as was previously shown for mammalian and yeast cells. Keeping

Appendix 3 L. Surmacz this in mind one might presume that all eukaryotic CPTs involved in dolichol chain synthesis require cooperation with NgBR homologues. 3. Identification of the bacterial-like trans/cis Pren-11 in S. cerevisiae and mammalian cells. Neither the enzymatic machinery responsible for its synthesis nor its role in the cells had been discovered. All eukaryotic CPTs described so far synthesize mixtures of polyisoprenoid homologues, therefore the mechanism of Pren-11 synthesis seems intriguing. Results of the studies summarized above raised several new questions which I would like to address in the future. Currently my goals are focused on: 1. Further studies on the role of the regulators of the CPT activity in polyisoprenoid biosynthesis and protein glycosylation in plants. (Project PRELUDIUM 8, leader Przemysław Surowiecki, PhD student working under my supervision); 2. Elucidation of the cellular role of eukaryotic Pren-11 in yeast and mammals: i) might it function as a glycosyl lipid-carrier in the protein glycosylation pathway similarly to Pren-11 in bacteria; ii) could it inhibit hydrolysis of intermediates of the protein glycosylation pathway (i.e. diphosphodolichyl-oligosaccharide, DLO) by inhibition of DLO diphosphatase activity (DLODP); iii) could it serve as a substrate for the biosynthesis of the longer polyisoprenoids; iv) does it protect cellular membranes against abiotic stress (These are the research tasks of my current project OPUS11).

Studies on biosynthesis of polyisoprenoids not only broaden the basic knowledge on metabolic homeostasis of the cell but in the long term they also open new perspectives for therapeutic interventions. In human, abnormal protein glycosylation leads to Congenital Disorders of Glycosylation (CDG) thus the obtained results might be helpful for designing therapeutic strategies for CDG patients.

In addition to the main line of studies described above, I was also involved in other projects focused on (i) vesicular transport in plants, (ii) analysis of the early stages of plant polyisoprenoid biosynthesis and (iii) conversion of polyprenol to dolichol in plants. The results of these studies are discussed below and are not a part of the achievement. In my opinion the approach linking these topics helps to understand the studied processes in the broader context and improves the effectiveness of studies on polyisoprenoid ‘superlipids’.

Appendix 3 L. Surmacz

5. Other scientific achievements:

My other scientific achievements, not included in the habilitation achievement, comprise 17 experimental papers, including 11 papers published prior to my PhD, 6 papers published in the postdoctoral period and 2 review papers.

Pre-doctoral achievements My initial project was focused on the search for a beta-adrenergic receptor (β-AR) in Paramecium aurelia and on studies of the effect of β-AR agonists and antagonists on endocytotic processes in this unicellular organism. Endocytosis was the main research topic of the Laboratory of Cell Membrane Physiology at the Nencki Institute for Experimental Biology. PAS, where I was working on my doctoral thesis under the supervision of Prof. Elzbieta Wyroba. The β-AR (β-AR1, -2 and -3) are G-coupled metabotropic receptors with seven transmembrane domains. Their stimulation activates adenylate cyclase, leading to increased level of cAMP in the cell. Prolonged activation of β1 and β2 receptors, but not β3, leads to their decreased sensitivities (desensitization). All β-AR are stimulated by endogenous catecholamines: noradrenaline and adrenaline. The presence of G-protein-coupled receptors and heterotrimeric G proteins involved in transduction of chemical and mechanical signals had been suggested earlier in Protozoa [Nakaoka et al., 1997; New et al., 2000; Marino et al., 2001]. Moreover, some unicellular eukaryotes were found to be sensitive to neurotransmitters [Roth et al., 1982; Nomura et al., 1998; Delmonte et al., 2001]. In unicellular organisms endocytosis serves as a food sourcing pathway. Endocytosis is subdivided into the following processes: i) phagocytosis – an uptake of large (food) particles (> 0.5 μm), e.g. bacteria; digestive vacuoles (phagosomes) are formed and next fused with lysosomes containing hydrolytic enzymes; ii) pinocytosis – an uptake of the fluid phase, in which large molecules (e.g. proteins and lipids) are dissolved; small vesicles called pinosomes are formed and next fused with lysosomes; iii) receptor-mediated endocytosis (RME) – allows the transport of specific compounds (e.g. hormones, growth factors) which are recognized by specific receptor proteins on the cell surface; it initiates the formation of clathrin-coated vesicles finally cleaved off by dynamin. In eukaryotic cells RME is critical for neurotransmission and signal transduction but not for supply of nutrients.

Using various techniques of molecular biology, we demonstrated in the Paramecium genome, which at that time had not been sequenced, the presence of DNA sequences homologous to the sequences encoding β-adrenergic receptor in multicellular organisms [Surmacz et al., Cell. Mol. Biol. Lett., 1997; Wiejak et al., Acta Protozool., 1998]. Confocal and electron microscopy studies revealed that the β-AR immunoanalogue is localized on the Paramecium cell surface and at the ridge of the cytopharynx, where 14

Appendix 3 L. Surmacz nascent phagosomes are formed, suggesting that the β-AR immunoanalogue appeared in this organism as an evolutionary ancient nutrient receptor. Treatment of Paramecium cells with a β-AR agonist, isoproterenol, resulted in enhanced phagocytic activity and altered distribution of the β-AR immunoanalogue in subcellular fractions - displacement of β-AR from membranous to cytosolic fraction might be correlated with receptor desensitization [Wiejak i in., Histochem. J., 2002]. Similar results had been obtained previously in mammalian cells [Mukherjee et al., 1975; Chuang and Costa, 1979; Zastrow and Kobilka, 1992]. Our observations were confirmed in imaging studies using confocal and electron microscopy. In the presence of isoproterenol, β-AR was translocated from the surface of the Paramecium cell into small vesicles in the cytoplasmic compartment. Additionally, we observed co- localization of β-AR immunoanalogue and dynamin in these structures. This suggested that in Paramecium, similarly to higher eukaryotes [Shetzline et al., 2002; Braun et al., 2003], agonist-mediated sequestration of β-AR is dynamin-dependent [Wiejak et al., J. Exp. Biol., 2004]. Moreover, we cloned Paramecium gene fragments homologous to the β-adrenergic receptor kinases 1 and 2, respectively β- ARK1=GRK2 [Wiejak et al., Histochem. J., 2002] and β-ARK2=GRK3 [Wiejak et al., J. Exp. Biol., 2004], which are involved in β-AR phosphorylation at the initial stage of receptor desensitization [Premont et al., 1995; Zastrow, 2001]. Our studies demonstrated that the molecular machinery responsible for the desensitization/sequestration of the receptor immune-related to vertebrate β-AR exists also in Paramecium cells. Further studies on phagocytosis in Paramecium showed complete absence of phagosomes in cells treated with propranolol (β1- and β2-adrenergic receptor blocker) or trifluoperazine (TFP, D2 - dopamine receptor blocker) indicating inhibition of phagocytic activity. In the cytosolic fractions isolated from such cells we observed a decrease of the intensity of three polypeptide bands. While the 75 and 87 kDa polypeptides were not identified, the 42 kDa polypeptide reacted with the antibody directed against actin. This result suggests that actin may be a target for pharmacological agents used in this study to inhibit Paramecium phagocytic activity [Surmacz et al., Folia Histochem. Cyto., 2001]. This result is consistent with the general opinion that all phagocytic processes are driven by a rearrangement of the actin cytoskeleton [May and Machesky, 2001]. Our subsequently performed studies revealed that dynamin is involved in Paramecium phagocytosis at its initial stage [Wiejak et al., Eur. J. Protistol., 2003]. In ciliates, phagosomes – also called digestive vacuoles, are formed due to fusions of discoidal vesicles with the cytopharyngeal membrane [Allen, 1974, Allen and Fok, 2000]. Ultrastructural analyses indicated that dynamin is localized at the cytopharyngeal membrane, in phagosomes and discoidal vesicles which are indispensable for phagosome membrane formation. In cells with inhibited phagocytic activity, we did not observe the presence of dynamin-positive discoidal vesicles. Stimulation of phagocytosis by feeding with polystyrene monodispersed latex beads evoked an increase of dynamin level compared to the control whereas phagocytosis inhibitors, propranolol or TFP – a decrease compared to the control [Wiejak et al., Eur. J. Protistol., 2003].

Appendix 3 L. Surmacz

We were the first to identify the presence of dynamin in ciliates. We cloned two dynamin encoding genes, ParD1 and ParD2, with high sequence homology to their mammalian counterparts. During the uptake of transferrin, the receptor-mediated endocytosis (RME) marker, which is transported into the cell by a dynamin- and clathrin dependent pathway, the dynamin level decreased by 25%. On the other hand, when RME was inhibited by ammonium chloride [Woods et al., 1989], the dynamin level in the cells decreased in a concentration-dependent manner. Using microscopic techniques we demonstrated the presence of dynamin in clathrin-coated vesicles during RME [Wiejak et al., Biochem. Cell Biol., 2004]. These results indicate that a dynamin- and clathrin-dependent RME pathway is present in Paramecium cells. In conclusion, we believe that dynamin is involved in analogous processes in Paramecium and mammalian cells: i) phagocytosis [Wiejak et al., Eur. J. Protistol., 2003], ii) agonist- induced receptor sequestration [Wiejak et al., J. Exp. Biol., 2004], and iii) receptor-mediated endocytosis [Wiejak et al., Biochem. Cell Biol., 2004]. Some reports postulated that pinosomes, vesicles formed during the fluid phase uptake, and endosomes ‘mix’ up inside the cell and consequently ligands internalized by both pathways are finally present in the same endosomes [Spiro et al., 1996; Ellinger et al., 1998; Synnes at al., 1999]. Our studies on pinocytosis and RME did not confirm such observations in Paramecium cells. Lucifer yellow (LY), a fluid phase tracer, administered simultaneously with transferrin, RME marker, did not co-localize (confocal microscopy image). LY showed (a typical for fluid phase uptake) diffused localization in the entire cell, whereas transferrin accumulated in the membrane-surrounded endosomes. Additionally, we measured the effect of RME stimulators (phorbol esters, PMA – acting via PKC activation) or inhibitors (wortmannin – PI-3K inhibitor or GF – PKC inhibitor) on LY accumulation in Paramecium cells. PMA was found to slightly reduce LY accumulation in the cells, while wortmannin and PKC inhibitor did not affect the fluid phase uptake by Paramecium. Taken together this suggests that PMA does not affect the fluid phase uptake via PKC [Wiejak et al., Eur. J. Histochem., 2001]. Our further studies suggest that the uptake of the fluid phase by Paramecium might be regulated through activation of cAMP-dependent kinase A (PKA). Studies on the effect of PKA inhibitor (iPKA) or PKA activator (IBMX) on fluid phase uptake were performed in the cells under conditions stimulating phagocytic activity. An increase of PKA activity did not change the accumulation of LY in the cells while its inhibition reduced the rate of fluid phase uptake by Paramecium [Wiejak et al., Folia Biol-Krakow, 2007]. Subsequently performed studies on regulation of endocytosis allowed us to identify the components of the machinery involved in endosome/phagosome formation, trafficking and vesicle transport in Paramecium. Vesicle transport is a selective and targeted type of transfer of substances enclosed inside vesicles formed from small membrane fragments. Numerous mechanisms of vesicular transport are common to all Eukaryotes. This type of transport is strictly regulated and controlled by Rab proteins of the family of small GTPases. Rab GTPs are involved in all stages of intracellular transport from vesicle formation through their transport to membranes of target compartment and fusion of membranes. Based

Appendix 3 L. Surmacz on the mammalian Rab protein sequence analysis, their counterparts in Paramecium cells were detected. Using various techniques of molecular biology (PCR, sequencing, Southern hybridization) we identified two homologues of the Rab7 protein, Rab7a and Rab7b, in Paramecium cells. Rab7 is localized in late endosomes in mammalian cells and regulates transport from the early to late endosomes and lysosomes thus playing a key role in the degradation of internalized substances [Feng et al., 1995] by controlling the rate of this process [Brucert et al., 2000]. Studies on Rab7 localization in Paramecium during endocytic processes revealed that Rab7 is present on the membranes of late endosomes during internalization of transferrin. Moreover, furin was also detected in this compartment. Furin recycles from late endosomes to TGN (trans Golgi network) in small transport vesicles where it co- localized with Rab7. Additionally, we identified the presence of furin in Paramecium (a fragment of the gene, respective mRNA and protein). Rab7 is present at the phagosome and primary lysosome (their fusion lead to phagolysosome formation) and secondary lysosome membranes during phagocytosis of polystyrene latex beads and bacteria. We demonstrated the co-localization of Rab7 and LAMP2 (lysosomal associated membrane protein 2), a typical protein of Metazoa lysosomes, in primary lysosomes and a LAMP2 protein homologue was identified in Paramecium cytosolic fraction. We also detected the presence of Rab7 effector protein – RILP (Rab-interacting lysosomal protein) in the lysosomal compartment. Co-localization of RILP and Rab7 in late endosomes and lysosomes suggests that both proteins play a role in vesicular transport between these compartments in Paramecium. Furthermore, two Rab7 isoforms are present in Paramecium late endosomes, phagosomes, primary and secondary lysosomes. This suggests that Rab7 plays a role in: i) transport between endosomes and lysosomes; ii) maturation of phagosomes and iii) control of fusion of lysosomes and phagosomes. The results presented here on Rab7, RILP and LAMP-2 were included in my PhD dissertation "Rab7 in Paramecium cells: studies on gene, protein and localization during endocytosis" and 2 experimental papers [Surmacz et al., Acta Biochim. Pol., 2006 and Wyroba et al., Eur. J. Histochem., 2007]. Our studies on the search for the homologues of the mammalian proteins in unicellular eukaryote Paramecium involved in endocytosis and membrane trafficking were summarized in the review “Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryotic Paramecium” [Surmacz et al., Biol. Cell, 2003].

In addition, during my employment at the Nencki Institute of Experimental Biology I had the opportunity to acquire expertise in the analysis of cell ultrastructure and protein immunolocalization with the aid of electron microscopy, and this expertise resulted in collaborative projects performed together with two research groups headed by Prof. Jacek Kuznicki and Prof. Adam Szewczyk. The results of these studies were described in two experimental papers [Filipek et al., J. Biol. Chem., 2002] and [Skalska et al., Biochim. Biophys. Acta, 2008].

Appendix 3 L. Surmacz

After finishing my PhD I successfully applied for a postdoctoral position at the Department of Lipid Biochemistry Institute of Biochemistry and Biophysics PAS. As a result I expanded my field of professional interest, acquired numerous new research methodologies and changed the experimental model. The knowledge and professional experience obtained during implementation of my doctoral project, however, provided a good starting point for these new challenges.

Post-doctoral achievements not included in the habilitation

During my employment as a post-doctoral fellow at the Department of Lipid Biochemistry Institute of Biochemistry and Biophysics of the Polish Academy of Sciences headed by Prof. Ewa Swiezewska I performed studies within the frame of the POL-POSTDOC II project entitled "The role of protein prenylation and prenylated lipids in the vesicular transport in plants”. The studies included two main research tasks: i) the role of Rab Escort Protein (REP) in prenylation of Rab proteins in plants and ii) studies on cis-prenyltransferases of Arabidopsis thaliana. The first part of this project concerning the vesicular transport regulated by Rab proteins, was closely related to my PhD project. Rab proteins are posttranslationally modified by covalent attachment of geranylgeranyl lipid groups which is a prerequisite of their binding to the membranes of intracellular compartments. Prenylation is catalyzed by a heterodimeric enzyme RabGGTase (Rab geranylgeranyl transferase) which requires an accessory subunit - REP (Rab Escort Protein). The role of the REP protein in plants was poorly characterized at that time and to achieve the goal of the project an Arabidopsis AtREP T-DNA insertion mutant (rep-/-), presumably devoid of functional RabGGTase, was used. I observed mis-localization of Rab proteins in rep-/- mutant: these proteins were mostly present in the cytosolic fraction whereas in wild type plants – in the membrane fraction; this confirmed disturbances in Rab geranylgeranylation in rep-/- lines. Furthermore, rep-/- mutant plants exhibited increased sensitivity to salinity. In vitro assay confirmed significantly decreased activity of RabGGTase in leaf extract from this mutant. This line of studies is currently continued by other researchers at the Department of Lipid Biochemistry, manuscripts describing these results are in preparation. The results of the experiments performed within the second part of my POL-POSTDOC project are described above as a part of the main scientific achievement.

During my employment at the IBB PAS, I was also involved in additional projects performed at the Department of Lipid Biochemistry, which addressed other topics in the field of polyisoprenoid biosynthesis and their role in plants. The results of these studies were described in three experimental papers:

Appendix 3 L. Surmacz

1. The role of polyisoprenoids in plant resistance to biotic stress was studied in tobacco plants Nicotiana tabacum cv. Samsun NN [Bajda et al., Physiol. Plant., 2009]. We found that polyisoprenoid alcohols and plastoquinone (PQ) are involved in local and systemic resistance of tobacco plants upon infection with avirulent pathogens, tobacco mosaic virus (TMV) or Pseudomonas syringae pv. Tabaci. Accumulation of PQ was also stimulated by hydrogen peroxide and salicylic acid (SA) whereas polyprenol production was stimulated by hydrogen peroxide. These results suggest that polyprenols accumulated in the cells play a role in defense responses to pathogens. Elevated activity of several antioxidant enzymes (ascorbate peroxidase, guaiacol peroxidase, glutathione reductase and superoxide dismutase, especially the CuZn superoxide dismutase isoform) and high, but transient elevation of catalase were found in inoculated leaves of resistant but not of susceptible tobacco plants.

2. Dolichols play a key role as cofactor of protein N-, O- and C-glycosylation in all eukaryotic organisms. Their biosynthesis involves the formation of polyprenyl diphosphate (Pren-PP) of the desired length performed by cis-prenyltransferase [CPT], subsequently Pren-PP is dephosphorylated [Frank and Waechter, 1998] and converted to dolichol by polyprenyl reductase (SRD5A3 in human) [Cantagrel et al., 2010]. The latter never been characterized in plants. We have identified and characterized two genes, PPRD1 and -2, orthologous to human SRD5A3 (steroid 5a reductase type 3) encoding polyprenol reductases responsible for conversion of polyprenol to dolichol in Arabidopsis thaliana [Jozwiak et al., Plant Cell, 2015]. PPRD1 and -2 play dedicated roles in plant metabolism. PPRD2 is essential for plant viability. Its deficiency results in aberrant development of the male gametophyte and sporophyte. Impaired protein glycosylation seems to be the major factor underlying these defects although disturbances in other cellular dolichol-dependent processes could also contribute. Shortage of dolichol in PPRD2-deficient cells is partially rescued by PPRD1 overexpression. Supplementation with dolichol (in vitro pollen germination test) also eliminates the phenotypic effects of dolichol deficiency. Identification of PPRD1 and -2 elucidates the factors mediating the key step of the dolichol cycle in plant cells which makes manipulation of dolichol content in plant tissues feasible.

3. Elucidation of cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors seems be indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Our recent paper, Jozwiak et al., Plant Physiol., 2017, describes a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols, designed to quantitatively follow the cooperation of the MVA and MEP pathways reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis roots. We found that the contribution of the MEP pathway was increased significantly (reaching 100%) exclusively for the dominating medium-chain dolichols (Dol-16), while for long-chain dolichols (Dol-21), the relative input of the MEP and MVA pathways remained unchanged, suggesting divergent sites of synthesis for medium- and long-chain dolichols. The analysis of numerically modeled dolichol mass

Appendix 3 L. Surmacz spectra is a novel method to follow modulation of the concomitant activity of isoprenoid-generating pathways in plant cells; additionally, it suggests an exchange of isoprenoid intermediates between plastids and peroxisomes.

Results published in the last two papers, Jozwiak et al., Plant Cell, 2015 and Jozwiak et al., Plant Physiol., 2017 were included in the doctoral dissertation of Adam Jozwiak - I was an assistant supervisor of this thesis.

In addition, I was invited to write a review manuscript on the biological role of plant polyisoprenoids, which comprises a part of the large monographic book "Isoprenoid Synthesis in Plants and Microorganisms. New Concepts and Experiments Approaches" edited by Thomas J. Bach and Michel Rohmer, Springer Science+Business Media New York 2013, pp. 307-313. In the chapter "What we do and do not know about the cellular functions of polyisoprenoids" [Surmacz and Swiezewska, 2013] we discussed the results of current studies in the context of membrane permeability and protein modification.

References

Akhtar TA, Matsuba Y, Schauvinhold I, Yu G, Lees HA, Klein SE, Pichersky E. (2013) The tomato cis-prenyltransferase gene family, Plant J. 73, 640-652. Akhtar TA, Surowiecki P, Siekierska H, Kania M, Van Gelder K, Rea K, Virta L, Vatta M, Gawarecka K, Wojcik J, Danikiewicz W, Buszewicz D, Swiezewska E, Surmacz L (2017) Polyprenols are synthesized by a plastidial cis- prenyltransferase and influence photosynthetic performance. Plant Cell 29, 1709-1725. Allen RD, Fok AK. (2000) Membrane trafficking and processing in Paramecium. Int. Rev. Cytol. 198, 277-318. Allen RD. (1974) Food vacuole membrane growth with microtubule-associated membrane transport in Paramecium. J. Cell Biol. 63, 904-922. Asawatreratanakul K, Zhang YW, Wititsuwannakul D, Wititsuwannakul R, Takahashi S, Rattanapittayaporn A, Koyama T. (2003) Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis, Eur. J. Biochem. 270, 4671-4680. Bajda A, Konopka-Postupolska D, Krzymowska M, Hennig J, Skorupinska-Tudek K, Surmacz L, Wójcik J, Matysiak Z, Chojnacki T, Skorzynska-Polit E, Drazkiewicz M, Patrzylas P, Tomaszewska M, Kania M, Swist M, Danikiewicz W, Piotrowska W, Swiezewska E. (2009) Role of polyisoprenoids in tobacco resistance against biotic stresses. Physiol. Plant. 135, 351-364. Brasher MI, Surmacz L, Leong B, Pitcher J, Swiezewska E, Pichersky E, Akhtar TA. (2015) A two-component enzyme complex is required for dolichol biosynthesis in tomato. Plant J. 82, 903-914. Braun L, Christophe T, Boulay F. (2003) Phosphorylation of key serine residues is required for internalization of the complement 5a (C5a) anaphylatoxin receptor via a beta-arrestin, dynamin, and clathrin-dependent pathway. J. Biol. Chem. 278,4277-4285. Bruckert F, Laurent O, Satre M. (2000) Rab7, a multifaceted GTP-binding protein regulating access to degradative compartments in eu karyotic cells. Protoplasma 210, 108-116. Burda P, Aebi M. (1999) The dolichol pathway of N-linked glycosylation. Biochim. Biophys. Acta 142, 239-257. Cantagrel V, Lefeber DJ, Ng BG, Guan ZQ, Silhavy JL, Bielas SL, Lehle L, Hombauer H, Adamowicz M, Swiezewska E, et al. (2010) SRD5A3 Is Required for Converting Polyprenol to Dolichol and Is Mutated in a Congenital Glycosylation Disorder. Cell 142, 203-217. Chuang DM, Costa E (1979) Evidence for internalization of the recognition site of β-adrenergic receptors during receptor subsensitivity induced by (-)-isoproterenol. Proc. Natl. Acad. Sci. USA 76, 3024-3028. Cunillera N, Arro M, Fores O, Manzano D, Ferrer A. (2000) Characterization of dehydrodolichyl diphosphate synthase of Arabidopsis thaliana a key enzyme in dolichol biosynthesis. FEBS Lett. 477, 170-174. 20

Appendix 3 L. Surmacz

Delmonte Corrado MU, Politi H, Ognibene M, Angelini C, Trielli F, Ballarini P, Falugi C. (2001) Synthesis of the signal molecule acetylcholine during the developmental cycle of Paramecium primaurelia (Protista, Ciliophora) and its possible function in conjugation. J. Exp. Biol. 204, 1901-1907. Denbow CJ, Lang S, Cramer CL. (1996) The N terminal domain of tomato 3-hydroxy-3-methylglutaryl-CoA reductases - Sequence, microsomal targeting, and glycosylation. J. Biol. Chem. 271, 9710-9715. Disch A, Schwender J, Muller C, Lichtenthaler HK, Rohmer M. (1998) Distribution of the mevalonate and glyceraldehyde phosphate/pyruvate pathways for isoprenoid biosynthesis in unicellular algae and the cyanobacterium Synechocystis PCC 6714. Biochem. J. 333, 381-388. Ellinger I, Klapper H, Fuchs R. (1998) Fluid-phase marker transport in rat liver: free-flow electrophoresis separates distinct endosome subpopulations. Electrophoresis 19, 1154-1161. Endo S, Zhang YW, Takahashi S, Koyama T. (2003) Identification of human dehydrodolichyl diphosphate synthase gene, Biochim. Biophys. Acta 1625, 291-295. Feng Y, Press B, Wandinger-Ness A. (1995) Rab 7: an important regulator of late endocytic membrane traffic. J. Cell Biol. 131, 1435-1452. Filipek A, Jastrzebska B, Nowotny M, Kwiatkowska K, Hetman M, Surmacz L, Wyroba E, Kuznicki J. (2002) Ca2+-dependent translocation of the calcyclin-binding protein in neurons and neuroblastoma NB-2a cells. J. Biol. Chem. 277, 21103-21109. Frank DW, Waechter CJ. (1998) Purification and characterization of a polyisoprenyl phosphate phosphatase from pig brain. Possible dual specificity. J. Biol. Chem. 273, 11791-11798. Fujihashi M, Zhang YW, Higuchi Y, Li XY, Koyama T, Miki K (2001) Crystal structure of cis-prenyl chain elongating enzyme, undecaprenyl diphosphate synthase. Proc. Natl. Acad. Sci. USA 98, 4337-4342. Grabińska KA, Cui J, Chatterjee A, Guan Z, Raetz CR, Robbins PW, Samuelson J. (2010) Molecular characterization of the cis- prenyltransferase of Giardia lamblia. Glycobiology 20, 824-832. Harrison KD, Miao RQ, Fernandez-Hernando C, Suarez Y, Davalos A, Sessa WC. (2009) Nogo-B receptor stabilizes Niemann- Pick type C2 protein and regulates intracellular cholesterol trafficking. Cell Metab. 10, 208-218. Harrison KD, Park EJ, Gao N, Kuo A, Rush JS, Waechter CJ, Lehrman MA, Sessa WC. (2011) Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation. EMBO J. 30, 2490-2500. Hemmerlin, A., Harwood, J.L., and Bach, T.J. (2012). A raison d'etre for two distinct pathways in the early steps of plant isoprenoid biosynthesis? Prog. Lipid Res. 51, 95-148. Hemming FW. (1983) The biosynthesis of polyisoprenoid chains. Biochem. Soc. Trans. 11, 497-504. Jozwiak A, Gutkowska M, Gawarecka K, Surmacz L, Buczkowska A, Lichocka M, Nowakowska J, Swiezewska E. (2015) POLYPRENOL REDUCTASE2 Deficiency Is Lethal in Arabidopsis Due to Male Sterility. Plant Cell 27, 3336-3353. Jozwiak A, Lipko A, Kania M, Danikiewicz W, Surmacz L, Witek A, Wojcik J, Zdanowski K, Pączkowski C, Chojnacki T, Poznanski J, Swiezewska E. (2017) Modelling of dolichol mass spectra isotopic envelopes as a tool to monitor isoprenoid biosynthesis. Plant Physiol. 174, 857-874. Jozwiak A, Ples M, Skorupinska-Tudek K, Kania M, Dydak M, Danikiewicz W, Swiezewska E. (2013) Sugar availability modulates polyisoprenoid and phytosterol profiles in Arabidopsis thaliana hairy root culture. Biochim. Biophys. Acta 1831, 438- 447. Kera K, Takahashi S, Sutoh T, Koyama T, Nakayama T. (2012) Identification and characterization of a cis, trans-mixed heptaprenyl diphosphate synthase from Arabidopsis thaliana. FEBS J. 279, 3813-3827. Kharel Y, Koyama T. (2003). Molecular analysis of cis-prenyl chain elongating enzymes. Nat. Prod. Rep. 20, 111-118. Ko TP, Chen YK, Robinson H, Tsai PC, Gao YG, Chen AP, Wang AH, Liang PH. (2001) Mechanism of product chain length determination and the role of a flexible loop in Escherichia coli undecaprenyl-pyrophosphate synthase catalysis. J. Biol. Chem. 276, 47474-47482. Kovacs WJ, Olivier LM, Krisans SK. (2002) Central role of peroxisomes in isoprenoid biosynthesis. Prog. Lipid Res. 41, 369- 391. Kwon M, Kwon EJ, Ro DK (2016) cis-Prenyltransferase and Polymer Analysis from a Natural Rubber Perspective. Methods Enzymol. 576, 121-45. Lange BM, Croteau R. (1999) Isopentenyl diphosphate biosynthesis via a mevalonateindependent pathway: Isopentenyl monophosphate kinase catalyzes the terminal enzymatic step. Proc. Natl. Acad. Sci. USA 96, 13714-13719. Lange BM, Ghassemian M. (2003) Genome organization in Arabidopsis thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism. Plant Mol. Biol. 51, 925-948. Lange BM, Wildung MR, McCaskill D, Croteau R. (1998) A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway. Proc. Natl. Acad. Sci. USA 95, 2100-2104.

Appendix 3 L. Surmacz

Lechner J, Wieland F, Sumper M. (1985) Biosynthesis of sulfated saccharides N-glycosidically linked to the protein via glucose. Purification and identification of sulfated dolichyl monophosphoryl tetrasaccharides from halobacteria. J. Biol. Chem. 260, 860- 866. Liang PH. (2009) Reaction Kinetics, Catalytic Mechanisms, Conformational Changes, and Inhibitor Design for Prenyltransferases. Biochemistry 48, 6562-6570. Lichtenthaler HK. (1999) The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 47-65. Liu MC, Wang BJ, Huang JK, Wang CS. (2011) Expression, Localization and Function of a cis-Prenyltransferase in the Tapetum and Microspores of Lily Anthers. Plant Cell Physiol. 52, 1487-1500. Marino MJ, Sherman TG, Wood DC. (2001) Partial cloning of putative G-proteins modulating mechanotransduction in the ciliate stentor. J. Eukaryot. Microbiol. 48, 527-536. May RC, Machesky LM. (2001) Phagocytosis and the actin cytoskeleton. J. Cell Sci. 114,1061-1077 Mukherjee C, Caron MG, Lefkowitz RJ (1975) Catecholamine-induced subsensitivity of adenylate cyclase associated with loss of β-adrenergic receptor binding sites. Proc. Natl. Acad. Sci. USA 72, 1945-1949 Nakaoka Y, Tanaka T, Kuriu T, Murata T. (1997) Possible mediation of G-proteins in cold-sensory transduction in Paramecium multimicronucleatum. J. Exp. Biol. 200, 1025-1030. New DC, Wong YH, Wong JT. (2000) Cloning of a novel G-protein-coupled receptor from the sea anemone nervous system. Biochem. Biophys. Res. Commun. 271, 761-769. Nguyen NQ, Lee S-C, Yang T-J, Lee OR. (2017) cis-Prenyltransferase interacts with a Nogo-B receptor homolog for dolichol biosynthesis in Panax ginseng Meyer. J. Ginseng Res. – in press doi.org/10.1016/j.jgr.2017.01.013 Nomura T, Tazawa M, Ohtsuki M, Sumi-Ichinose C, Hagino Y, Ota A, Nakashima A, Mori K, Sugimoto T, Ueno O, Nozawa Y, Ichinose H, Nagatsu T. (1998) Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 120, 753-760. Nothaft H, Szymanski CM. (2010) Protein glycosylation in bacteria: sweeter than ever. Nat. Rev. Microbiol. 8, 765-778. Oh SK, Han KH, Ryu SB, Kang H. (2000) Molecular cloning, expression, and functional analysis of a cis-prenyltransferase from A. thaliana. Implications in rubber biosynthesis. J. Biol. Chem. 275, 18482-18488. Park EJ, Grabińska KA, Guan Z, Stránecký V, Hartmannová H, Hodaňová K, Barešová V, Sovová J, Jozsef L, et al. (2014) Mutation of Nogo-B receptor, a subunit of cis-prenyltransferase, causes a congenital disorder of glycosylation. Cell Metab. 20, 448-457. Pattison RJ, Amtmann A. (2009) N-glycan production in the endoplasmic reticulum of plants. Trends Plant Sci. 2, 92-99. Premont RT, Inglese J, Lefkowitz RJ (1995) Protein kinases that phosphorylate activated G protein-coupled receptors. FASEB J. 9, 175-182. Proteau PJ. (1998) Biosynthesis of phytol in the cyanobacterium Synechocystis sp. UTEX 2470: Utilization of the non- mevalonate pathway. J. Nat. Prod. 61, 841-843. Qu Y, Chakrabarty R, Tran HT, Kwon EJ, Kwon M, Nguyen TD, Ro DK. (2015) A lettuce (Lactuca sativa) homolog of human Nogo-B receptor interacts with cis-prenyltransferase and is necessary for natural rubber biosynthesis. J. Biol. Chem. 290, 1898- 1914. Rohmer M, Knani M, Simonin P, Sutter B, Sahm H. (1993) Isoprenoid biosynthesis in bacteria – a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem. J. 295, 517-524. Rossignol DP, Scher M, Waechter CJ, Lennarz WJ. (1983) Metabolic interconversion of dolichol and dolichyl phosphate during development of the Sea urchin embryo. J. Biol. Chem. 258, 9122-9127. Rush JS, Matveev S, Guan Z, Raetz CR, Waechter CJ. (2010) Expression of functional bacterial undecaprenyl pyrophosphate synthase in the yeast rer2{Delta} mutant and CHO cells. Glycobiology 20, 1585-1593. Sato M, Fujisaki S, Sato K, Nishimura Y, Nakano A. (2001) Yeast Saccharomyces cerevisiae has two cis-prenyltransferases with different properties and localizations. Implication for their distinct physiological roles in dolichol synthesis, Genes Cells 6, 495-506. Sato M, Sato K, Nishikawa S, Hirata A, Kato J, Nakano A. (1999) The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cis-prenyltransferase, a key enzyme in dolichol synthesis, Mol. Cell. Biol. 19, 471-483. Schmidt T, Lenders M, Hillebrand A, van Deenen N, Munt O, Reichelt R, Eisenreich et al. (2010) Characterization of rubber particles, rubber chain elongation in Taraxacum koksaghyz, BMC Biochem. 11,11. Shetzline MA, Walker JK, Valenzano KJ, Premont RT. (2002) Vasoactive intestinal polypeptide type-1 receptor regulation. Desensitization, phosphorylation, and sequestration. J. Biol. Chem. 277, 25519-25526.

Appendix 3 L. Surmacz

Shimizu N, Koyama T, Ogura K. (1998) Molecular cloning, expression, and purification of undecaprenyl diphosphate synthase. No sequence similarity between E- and Z-prenyl diphosphate synthases, J. Biol. Chem. 273, 19476-19481. Shridas P, Rush JS, Waechter CJ. (2003) Identification and characterization of acDNA encoding a long-chain cis- isoprenyltranferase involved in dolichylmonophosphate biosynthesis in the ER of brain cells. Biochem. Biophys. Res. Commun. 312, 1349-1356. Skalska J, Piwońska M, Wyroba E, Surmacz L, Wieczorek R, Koszela-Piotrowska I, Zielińska J, Bednarczyk P, Dołowy K, Wilczynski GM, Szewczyk A, Kunz WS. (2008) A novel potassium channel in skeletal muscle mitochondria. Biochim. Biophys. Acta 1777, 651-659. Skorupinska-Tudek K, Poznanski J, Wojcik J, Bienkowski T, Szostkiewicz I, Zelman-Femiak M, Bajda A, Chojnacki T, Olszowska O, Grunler J, Meyer O, Rohmer M, Danikiewicz W, Swiezewska E. (2008). Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants. J. Biol. Chem. 283, 21024-21035. Smit A, Mushegian A. (2000) Biosynthesis of isoprenoids via mevalonate in archaea: The lost pathway. Genome Research 10, 1468-1484. Spiro DJ, Boll W, Kirchhausen T, Wessling-Resnick M. (1996) Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro. Mol. Biol. Cell 7, 355-367. Surmacz L, Krawczyk K, Wyroba E. (1997) Different beta-adrenergic-specific molecular probes reveal the same DNA species in Paramecium hybridization analysis. Cell. Mol. Biol. Lett. 2, 379-387. Surmacz L, Swiezewska E. (2011) Polyisoprenoids - Secondary metabolites or physiologically important superlipids? Biochem. Biophys. Res. Commun. 407, 627-632. Surmacz L, Wiejak J, Wyroba E. (2001) Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis. Folia Histochem Cytobiol. 39, 301-305. Surmacz L, Wiejak J, Wyroba E. (2003) Evolutionary conservancy of the endocytic and trafficking machinery in the unicellular eukaryote Paramecium. Biol. Cell. 95, 69-74. Surmacz L, Wiejak J, Wyroba E. (2006) Cloning of two genes encoding Rab7 in Paramecium. Acta Biochim. Pol. 53, 149-156. Surmacz L, Plochocka D, Kania M, Danikiewicz W, Swiezewska E. (2014) cis-Prenyltransferase AtCPT6 produces a family of very short-chain polyisoprenoids in planta. Biochim. Biophys. Acta. 1841, 240-250. Surmacz L, Wojcik J, Kania M, Bentinger M, Danikiewicz W, Dallner G, Surowiecki P, Cmoch P, Swiezewska E. (2015) Short-chain polyisoprenoids in the yeast Saccharomyces cerevisiae - New companions of the old guys. Biochim. Biophys. Acta 1851, 1296-1303. Swiezewska E, Thelin A, Dallner G, Andersson B, Ernster L. (1993) Occurrence of prenylated proteins in plant cells. Biochem. Biophys. Re.s Commun. 192, 161-166. Swiezewska E and Danikiewicz W. (2005) Polyisoprenoids: Structure, biosynthesis and function. Prog. Lipid Res. 44, 235-258. Synnes M, Prydz K, Lovdal T, Brech A, Berg T. (1999) Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes. Biochim. Biophys. Acta 1421, 317-328 Wang C, Chen Q, Fan D, Li J, Wang G, Zhang P.(2016) Structural Analyses of Short-Chain Prenyltransferases Identify an Evolutionarily Conserved GFPPS Clade in Brassicaceae Plants. Mol. Plant. 9, 195-204. Wang W, Dong C, McNeil M, Kaur D, Mahapatra S, Crick DC, Naismith JH. (2008) The structural basis of chain length control in Rv1086. J. Mol. Biol. 381, 129-140. Wiejak J, Platek A, Surmacz L, Wyroba E. (1998) PCR amplification of Paramecium DNA using the beta-adrenergic- specific primers Acta Protozool. 37, 215-219. Wiejak J, Surmacz L, Wyroba E. (2001) Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin. Eur. J. Histochem.45, 383-388. Wiejak J, Surmacz L, Wyroba E. (2002) Immunoanalogue of vertebrate beta-adrenergic receptor in the unicellular eukaryote Paramecium. Histochem. J. 34, 51-56. Wiejak J, Surmacz L, Wyroba E. (2003) Dynamin involvement in Paramecium phagocytosis. Eur. J. Protistol. 39, 416- 422. Wiejak J, Surmacz L, Wyroba E (2004) Dynamin- and clathrin-dependent endocytic pathway in unicellular eukaryote Paramecium. Biochem. Cell Biol. 82, 547-558. Wiejak J, Surmacz L, Wyroba E. (2004) Dynamin-association with agonist-mediated sequestration of beta-adrenergic receptor in single-cell eukaryote Paramecium. J. Exp. Biol. 207, 1625-1632. Wiejak J, Surmacz L, Wyroba E. (2007) Pharmacological Attenuation of Paramecium Fluid-Phase Endocytosis. Folia Biol-Krakow 55, 95-100.

Appendix 3 L, Surmacz

\Mlding El, Brown JR, Bryant AP, Chalker AF, Holmes DJ, lngraham l(A, lordanescu S, Chi YS, Rosenberg M, Gwynn MN. (2000) ldentification, evolution, and essentiality of the mevalonate pathway for isopentenyl diphosphate biosynthesis in gram_ positive corci. J. Bacteriol. 182, 43194327. Wolucka BA, McNeil MR, de Hoffrnann E, Chojnacki T, Brennan PJ. (1994) Recognition of the lipid intermediate for arabinogalactan/arabinomannan biosynthesis and its relation to the mode of action of ethambutol on mycobacteria. J. Biol. Chem. 269, 23328-23335. Woods, J.W., Goodhouse, J., and Farquhar, M.G. 1989. Transferrin receptors and cation-independent mannose€-phosphate receptors deliver their ligands to two distinct subpopulations of multivesicular endosomes. Eur. J. Cell Biol. 50, 132-143. Wyroba E, Surmacz L, Osinska il, Wieiak J. (2007) Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues. Eur. J. Histochem. 51, 163-172. Zastrow M (2001) Role of endocytosis in signalling and regulation of G-protein-coupled receptors. Biochem Soc Trans 29, 500_ 504.

Zastrow M, Kobilka BK (1992) Ligand-regulated internalization and recycling of human B2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J. Biol. Chem. 267, 3530-3538. Zhang H, Ohyama K, Boudet J, Chen Z, Yang J, Zhang M, Muranaka T, Maurel C, Zhu JK, Gong Z. (2008) Dolichol biosynthesis and its effec§ on the unfolded protein response and abiotic stress resistiance in Arabidopsis, Plant Cell 20, 1879_ 1898.

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