Biochemical characterization of a novel lysosomal membrane protein disrupted in renal carcinoma 2 (DIRC2) [Elektronische Ressource] / submitted by Lalu Rudyat Telly Savalas

Biochemical Characterization of a Novel
Lysosomal Membrane Protein
Disrupted in Renal Carcinoma 2 (DIRC2)



Dissertation
in fulfillment of the requirement for the degree “Dr. rer. nat”
of the Faculty of Mathematics and Natural Sciences
at Kiel University











submitted by
Lalu Rudyat Telly Savalas
Kiel 2011











Referees:
1. Prof. Dr. Paul Saftig
2. Prof. Dr. Matthias Leippe

Date of oral examination : 13.05.2011
Approved for publication : 24.05.2011

Signed: Dean



Herewith I declare that:
1. Apart from the supervisor`s guidance, the contents and design of this dissertation
are my own work.
2. This thesis has not been in partially or wholly as part of a doctoral degree to
another examining body.
Parts of this work have been submitted for scientific publication
3. This thesis has been prepared according to the Rules of Good Scientific Practice of
the German Research Foundation.







Kiel, March 9, 2011


Lalu Rudyat Telly Savalas







Acknowledgement

I would like to thank to Prof. Dr. Paul Saftig and Dr. Bernd Schroeder who guided me
throughout my PhD thesis.
I would like to express my grateful to the Saftig´s present and former members: Eeva-Liisa
Eskelinen, Karine Reiss, Seenu, Beimi, Marc, Alex, Christina Wehling, Marion, Jenny, Kathi,
Micha, Judith, Marlies, Inez, Fr. Z, Michelle, Johann, Janna, Sebastian, Raffi, Andrea, Nur,
Jockel, Hannes, Silvio, Christian Raab, Mirka.
I would especially thank to the DAAD (Deutsher Akademischer Austaushdienst), the
Directorate of Higher Education, Republic of Indoensia, the Rector of University of Mataram
and the Dean of the Faculty of Teacher Training and Education, University of Mataram,
Indonesia for facilitating my study in Germany.
I am heartily indebted by the encouragement from my former teachers and lecturers: pak
Supardi, Dr. Dessy Natalia.
Thank you to the former and present PPI Kiel colleagues.
Thank you to Ibunda Baiq Nai´mah and Siti Syamsiyah serta Ayahanda Ahmad Ramli and
Sri Bintoro Hadiwidjojo, to my brothers and sisters Rully el Faraby, Very el Viera, Yudi
Islami Firdaus, Mba Uun, Mba Fivi, Dek Hudaya, Dek Ibadurrahman, Dek Hanif.
I would like to thank to my lovely wife Jannatin Àrdhuha and our sons Mukhlis and Hasan
Abdulhaq, who fueled me up to accomplish this work.


Table of contents
Table of Contents i
List of Figures iv
List of Tables vi
Abbreviations vii
Summary x
Zusammenfassung xii
1 Introduction …………………………………………………………… 1
1.1 Lysosomes ……………………………………………………………… 1
1.2 Targeting of lysosomal proteins .................................................................. 1
1.3 Soluble lysosomal proteins ……………………………………………… 6
1.4 Lysosomal membrane proteins …………………………………………… 9
1.4.1 Proteomic study of lysosomal membrane proteins ...................................... 9
1.4.2 Lysosomal transporter proteins 10
1.4.3 Disrupted in Renal Carcinoma 2 (DIRC2) ……………………………… 12
1.5 Major Facilitator Superfamily…………………… 13
1.6 Objectives …………………………………………………........................ 15
2 Materials and Methods ………………………………………………..... 16
2.1 Materials ……………………………………………….............................. 16
2.1.1 Equipments ……………………………………………….......................... 16
2.1.2 Disposable materials ………………………………………………............ 18
2.1.3 Cells ………………………………………………..................................... 18
2.1.4 Chemicals ................. 19
2.1.5 Plasmids ……………………………………………................................... 20
2.1.6 Primers and oligonucleotides …………………………………………….. 22
2.1.7 Constructs ……………………………………………................................ 22
2.1.8 Antibodies 23
2.1.9 Media ……………………………………………....................................... 24
2.1.10 Kits …………………………………………….......................................... 25
2.1.11 Buffers ……………………………………………..................................... 25
2.1.12 Enzymes …………………………….................................. 29
i
2.1.13 Softwares and web-based tools ………………………………………… 29
2.2 Methods ………………………………………………............................... 30
2.2.1 Molecular biology ……………………………………………................... 30
2.2.1.1 Basic PCR ……………………………………………................................ 30
2.2.1.2 Fusion PCR …………………………………………….............................. 31
2.2.1.3 Reverse transcription PCR ……………………………………………...... 33
2.2.1.4 DNA isolation …………………………………………….......................... 33
2.2.1.5 Agarose gel electrophoresis ……………………………………………..... 35
2.2.1.6 Gel elution ……………………………………………............................... 36
2.2.1.7 Determination of nucleic acid concentration …………………………… 36
2.2.1.8 DNA sequencing ……………………………………………..................... 36
2.2.1.9 RNA isolation 37
2.2.1.10 DNA digestion by restriction enzyme …………………............................. 37
2.2.1.11 Ligation …………………………………………….................................... 38
2.2.1.12 Dephosphorylation …………………………………………….................. 39
2.2.2 Biochemistry ……………………………………………............................ 39
2.2.2.1 Total lysate preparation ……………………………………………........... 39
2.2.2.2 Membrane protein preparation …………………………………………… 40
2.2.2.3 Protein determination …………………………………………….............. 40
®2.2.2.4 Percoll fractionation …………………………………………….............. 40
2.2.2.5 SDS-PAGE …………………………………………….............................. 41
2.2.2.6 Western blot ……………………………………………............................. 42
2.2.2.7 Enzyme assays …………………………………………............................. 44
2.2.2.8 Immunofluorescence …………………………………………................... 45
2.2.3 Cell biology …………………………………………................................. 46
2.2.3.1 Preparation of electro-competent E. coli ………………………………. 47
2.2.3.2 E. coli transformation ………………………………………….................. 47
2.2.3.3 DNA transfection …………………………………………......................... 48
2.2.3.4 siRNA transfection …………………………………………...................... 48
2.2.3.5 Protease inhibition in vivo …………………………………………........... 49
3 Results …………………………………………......................................... 50
3.1 Analysis of DIRC2 sequence …………………………………………… 50
3.2 DIRC2 is proteolytically processed ……………………………………… 53
ii
3.3 DIRC2 is a lysosomal membrane protein ………………………………… 53
3.4 DIRC2 is N-glycosylated at Asn-209 …………………………………… 56
3.5 Lysosomal targeting of DIRC2 depends on a lysosomal sorting motif at
its N-terminus ………………………………………….............................. 58
3.5.1 Expression of dileucine and ER retention mutants of DIRC2 …………… 58
3.5.2 Stepwise post-translational modification of DIRC2 .................................... 59
3.6 Detection of endogenous DIRC2 ………………………………………… 62
3.7 Inhibitory profiling indicates involvement of the lysosomal cysteine
protease cathepsin L …………………………………………................. 64
3.8 Processing of DIRC2 is abolished in Cathepsin L deficient fibroblasts … 66
3.9 Rescue of DIRC2 proteolysis by coexpression of cathepsin L in cathepsin
deficient MEF ………………………………………….............................. 70
3.10 Determination of the DIRC2 cleavage site ……………………………… 71
3.11 Overexpression of GFP-tagged DIRC2 and formation of
enlarged/clustered lysosomes …………………………………………...... 74
3.11.1 Overexpression of DIRC2 in mammalian cells lead to the formation of
enlarged and/or clustered of acidic compartments ……………………… 74
3.11.2 Effects of bafilomycin A1, vinblastine and Rab5 on the DIRC2-GFP
driven enlargement of acidic organelles …………. 74
4 Discussion …………… ……………………………................................... 78
4.1 DIRC2 is a novel lysosomal protein ……………………………………… 78
4.2 DIRC2 is a glycoprotein ………………………………………….............. 79
4.3 DIRC2 is proteolytically processed …………………………………….. 80
4.4 Cathepsin L is involved in the processing of DIRC2 81
4.5 Cleavage site of DIRC2 …………………………………………............... 83
4.6 Putative function of DIRC2 …………………………………………......... 85
4.7 Outlook …………………………………………........................................ 86
Bibliography ………… …………………………....................................... 88
Curriculum Vitae …… ………………………………............................... 98



iii
List of Figures

Figure 1.1 Schematic description of lysosomal system ………………………….. 2
Figure 1.2 Targeting of lysosomal proteins ……………………………………… 3
Figure 2.1 pEGFP-N1 ……………………………………………………………. 20
Figure 2.2 peGFP-C1 ……………………………………………………………. 21
Figure 2.3 pcDNA3.1/Hygro+ …………………………………………………… 21
Figure 2.4 Fusion PCR strategy ………………………………………………….. 32
Figure 2.5 Semi-dry transfer of protein ………………………………………….. 43
Figure 3.1 Proposed topology of human DIRC2…………………………………. 51
Figure 3.2 Expression of GFP and 3xmyc-tagged DIRC2 analyzed by Western
blot …………………………………………………………………… 52
Figure 3.3 Localization of DIRC2 ……………………………………………….. 54
®Figure 3.4 Percoll fractionation of the extract of HeLa cells transiently
transfected with DIRC2-3