Identification of Atg11p binding-partners involved in macropexophagy in the methylotrophic yeast Hansenula polymorpha
Subtitle
Determination and identification of unknown binding partners of atg11p involved in peroxisome degradation
Abstract
Defects in peroxisome biogenesis can cause degradation of peroxisomes that can lead to severe diseases in man that in some cases are lethal like the Zellweger syndrome. Degradation of peroxisomes is regulated by a pathway called macropexophagy. One of the proteins that is involved in this pathway is Autophagy-related protein 11 (Atg11p). Atg11 proteins accumulates in the vacuolar membrane region, where the peroxisomes contact the vacuole, by working as a scaffold protein that recruit ATG-proteins to the pre-autophagosome (PAS), the site of vesicle/autophagosome formation This protein recruits other Atg proteins by protein-protein interaction, that are also involved in this pathway, which most of them are still undiscovered. This research is focussed on the identification of the binding-partners of Atg11p in the methylotrophic yeast Hansenula polymorpha. The yeast was successfully grown on methanol as a sole carbon source and glucose-induced peroxisome degradation by macropexophagy was acquired. Co-immunoprecipitation was used to isolate any protein-complex involving Atg11p. The potentially associated proteins were identified by MALDI-TOF MS mass fingerprinting. A 74 kDa product was found after co-immunoprecipitation on SDS-PAGE. After mass fingerprint identification the product turned out to be alcohol oxidase. Although alcohol oxidase is peroxisome associated, involving lack of confidential results, it is probably not an interaction partner of Atg11p. Any affinity between Atg11p and alcohol oxidase could be an interesting subject for further research.
List with abbreviations and symbols
Abbreviations or symbols Term
AOX Alcohol oxidase
Atg11p Autophagy-related protein 11
Cvt Cytoplasm to vacuole transport
GFP Green Fluorescent Protein
PAS Pre-autophagosomale structure
PDD18 (syn. Atg11) Peroxisome degradation deficient protein 18
PMF Peptide Mass Fingerprinting
PAGE Poly-Acrylamide Gel Electrophoresis
SDS Sodium Dodecyl Sulfate
1. Introduction
Peroxisomes are essential organelles of eukaryotic cells. Peroxisomes function in many metabolic pathways, including β-oxidation of very long chain fatty acids and biosynthesis of plasmalogens. The molecular mechanism of peroxisome biogenesis is conserved among eukaryotic cells from yeasts to humans. However, the defects in peroxisome biogenesis cause severe diseases in man that in some cases are lethal like the Zellweger syndrome [1].
When peroxisomes have become dysfunctional or redundant, they are selectively degraded by the autophagic pathway. This removal of peroxisomes occurs via highly selective processes designated pexophagy. This autophagy-related pathway, occurs in a range of organisms from unicellular eukaryotes to mammals but has been studied the most in methylotrophic yeasts [2]. Growing these yeast cells on methanol as a carbon and energy source induces peroxisomes and the synthesis of peroxisomal alcohol oxidase (AOX) and other enzymes required for methanol metabolism (fig.2).
Figure 2: Methanol metabolism pathway in O. polymorpha (modified after Gellissen et al. 2005). 1 – alcohol oxidase, 2 – catalase, 3 – dihydroxyacetone synthase, 4 – formaldehyde dehydrogenase, 5 – formate dehydrogenase, 6 – dihydroxyacetone kinase, 7 – GSH – glu
In H. polymorpha, massively induced peroxisomes of methanol-grown cells are selectively degraded by macropexophagy when cells are shifted to glucose medium. During glucose-induced macropexophagy the organelles are characteristically sequentially degraded. In particular, large, mature organelles are degraded whereas at least one relatively small organelle escapes this process. In addition to ATG genes that encode components of the general autophagy machinery, specific proteins are also required for pexophagy [3].
One of these specific protein that’s involved in both is Atg11 p. Atg11 was originally identified as a component specifically required for the Cvt pathway and pexophagy, the latter defining the pathway for the selective degradation of peroxisomes. Atg11 proteins accumulates in the vacuolar membrane region, where the peroxisomes contact the vacuole, by working as a scaffold protein that recruit ATG proteins to the pre-autophagosome (PAS), the site of vesicle/autophagosome formation [4].
Deletion of ATG11 causes a defect in the localization of other Atg components to the PAS similar to the effects of eliminating the Cvt complex (Shintani et al., 2002). Because Atg11 can interact with the C- terminus of Atg19, these results suggest that Atg11 has some critical role in recruiting the Cvt complex to the PAS. But the regulatory mechanism of its multiple function, by which it recruits the Cvt complex to the PAS and selective peroxisome degradation is still unclear [5].
To understand the regulatory mechanism of Atg11p both in the Cvt-pathway and Macr0pexophagy, are we interested in finding the proteins that is helping Atg11p in these pathways.
In this research we focus on the macropexophagy pathway and investigate Atg11p binding-partners by using the biochemical techniques; Co-immunoprecipitation, SDS PAGE and mass spectrometry.
1.1 Techniques
Sample preparation
Three H. polymorpha strains (Veenhuis et al, University of Groningen), shown in table 1 are used for this research because of their ability to grow on methanol.
Table 1: H. polymorpha strains used for this research
Strain Definition Purpose
NCYC495 (leu1.1.) (WT) Leucine auxotrophic strain
Wild-type Used for molecular genetic purposes (disruption / deletion /
overexpression etc.)
Pdd18.GFP Peroxisome degradation deficiënt 18/Atg11p fused with GFP Atg11p
Hf206-SKL.GFP Peroxisomal matrix marker GFP-SKL Peroxisomal matrix marker GFP-SKL under the control of the substrate-inducible amine oxidase promoter (PAMO) in order to allow specific analysis of the organelle population present prior to the shift.
Co-immunoprecipitation (Co-IP)
Co-IP is used for protein-protein interaction identification and validation. The principle of the co-IP technique is immunoprecipitation of intact protein complexes. This works by selecting an antibody that targets a known protein that is believed to be a member of a larger complex of proteins. By targeting this known member with an antibody it may become possible to pull the entire protein complex out of solution and thereby identify unknown members of the complex (fig.3).
Figure 3: The principle of the co-IP technique is as follows: Many intracellular protein-protein interactions are retained when cells are lysed under non-denaturing conditions. The bait protein, for example, protein X, can be captured by its specific antibody stabilized to agarose beads. If there is another protein, the prey protein, for example, protein Y, binds to protein X in vivo, the protein X – protein Y complex can then be precipitated together by the antibody. Subsequently, through the investigation of protein Y, we can confirm the protein X – protein Y interaction, or discover new interactors of protein X (http://www.profacgen.com/Co-Immunoprecipitation-Co-IP.htm)
SDS Polyacrylamide Gel Electrophoresis
The unknown protein that is interacted with the known protein will be separated based on their charge and size by SDS PAGE (Polyacrylamide Gel Electrophoresis).
Peptide Mass Fingerprinting
PMF is used for the identification of the unknown protein that is interacted with known protein. Two dimensional electrophoresis separations generate patterns of protein spots that can be visualized and compared by image analysis. The proteins of interest are then cut from the gel. The gel pieces are destained, subjected to an in-gel digest and the peptides extracted and cleaned, as illustrated in figure 4a. The protein can then be identified by comparing the digest peaks with a computer-generated database of tryptic peptides (MASCOT) from known proteins (fig.4b).
Figure 4: A. Preparation of protein spots for MS identification, B. Identification by peptide mass fingerprinting
1.2 Objective of research
The aim of this research is to identify the binding-partner(s) of Atg11p that are involved in the metabolic pathway macropexophagy in the methylotrophic yeast Hansenula polymorpha.
1.2.1 Hypothesis
The expected outcomes in the various steps of the identification of Atg11p binding-partners are:
– Fluorescence microscopy of macropexophagy induced cells
o Wild-Type (negative control)
Wild-type cells will show no GFP expression.
o Hf206-GFP.SKL
Peroxisomal matrix marker GFP-SKL under the control of the substrate-inducible amine oxidase promoter (PAMO) is used for specific analysis of the organelle population presence. It will show many spots of peroxisome localization by expression of GFP fused to peroxisomal matrix.
o Pdd18 (Peroxisome degradation deficient Atg11) GFP
It will show few spots of Atg11 protein by expression of GFP fused with Atg11p located in the cytosol.
– SDS PAGE
o Known Protein
o GFP
26,9 kDa
o Atg11p H. polymorpha
149,18 kDa
o Atg11p fused with GFP
26,9 + 149,18 = 176,08 kDa
o Strain
– Wild-Type (negative control)
Precipitated wild-type cell lysate will show no protein bands on the gel.
– Hf206-GFP.SKL
Precipitated wild-type cell lysate will show no protein bands on the gel.
– Pdd18 (Peroxisome degradation deficient Atg11) GFP
Precipitated cell lysate with Atg11p fused with GFP will show a protein band of:
• ~ 176 kDa, if Atg11p didn’t interact/doesn’t have a binding partner.
• > 176 kDa, if Atg11p interact with a binding partner.
2. Material & Method
2.1 Micro-organisms and growth conditions.
In order to induce macropexophagy were the H. polymorpha strains, that were used in this study (listed in table 1), grown at 37 °C in complex media (YPD) containing 1% (w/v) yeast extract, 1% (w/v) peptone and 1% (w/v) glucose. For the proliferation of peroxisomes, were the cells subsequently diluted to 0.1 OD600 in fresh 0.5% glucose mineral media (MM) (van Dijken et al., 1976) with rich vitamin solution and Vishniac solution grown for two to three cell divisions (4-6 h). For peroxisome induction, cells were transferred to mineral media (MM) with 0.4% methanol as carbon source, supplemented with 30 mg/l leucine at a 1/10 overnight dilution. Macropexophagy was induced when 0,5% glucose was added to cells.
2.2 Microscopy
Fluorescence microscopy was used in order to analyse the organelle population present prior to the shift. For live-cell imaging, cells were transferred to an objective containing 1% agarose, incubated in the appropriate medium and kept at 37°C; 10% LED power was used for sequential imaging.
2.3 Preparation of protoplasts
Protoplasts were prepared in order to perform Co-immunoprecipitation. All cells were harvested by centrifugation 5 minutes 3800 x g at room temperature and weight of the pellets were determined. Cells were resuspended (~0.6 g/ml) in pre-incubation buffer (100 mM Tris.HCl pH 8.0, 50 mM EDTA) with 140 mM β-mercaptoethanol and incubated for 15 minutes at 35 °C while shaking. Cells were collected by centrifugation 5 minutes 3800 x g at room temperature and washed once in protoplast buffer (50 mM Kpi (pH 7.2), 1.2 M sorbitol). Protoplasts were prepared by treatment with zymolase (MP Biomedicals) in protoplast buffer (50 mM Kpi (pH 7.2), 1.2 M sorbitol, zymolase (1 mg/ml) for 1 h in a water bath at 35 °C while shaking (50 rpm).
2.4 Co-immunoprecipitation (Co-IP)
Co-IP was performed in order to precipitate the protein-complex of Atg11p.GFP-unknown protein. All protoplasts were harvest by centrifugation (13 000 rpm, 10 min, 4 °C) and resuspended in lysis buffer (150 mM KCl, 20 mM HEPES (pH 7.4), 2 mM DTT, 2 mM EDTA, one complete protease inhibitor cocktail tablet/25 ml (Roche), 4 mM MgCl, +/- 0.5 mM ATP, 0.5% Triton X-100). Whole cell lysates were obtained by Potter-Elvehjem homogenisation followed by 10 min incubation on ice with addition of 0.5% (v/v) CHAPS. After centrifugation (13 000 g, 5 min, 4 °C), protein concentration was determined with a BCA kit (Pierce BCA Protein Assay Kit) and lysates were incubated by rocking with protein A-Sepharose (Sigma-Aldrich; 80 μL; pre-equilibrated in lysis buffer) at 4 °C overnight with antibody anti-GFP. Sepharose beads were harvested by sedimentation and washed two times with lysis buffer containing 0.1% (v/v) Triton X-100. The bound material was eluted by addition of lysis buffer and boiling with 5x SDS PAGE buffer (containing β-mercaptoethanol, SDS, glycerol, TRIS-HCl and bromophenol blue) at 95 °C for 5 minutes.
2.5 Electrophoresis materials and instrumentation.
Electrophoresis cells, polyacrylamide gels and reagent for gel electrophoresis (MOPS running buffer, loading buffer, reducing agent) were purchased from Invitrogen and ZipTip pipette tips for microvolume sample preparation of peptides prior to (MALDI-TOF) mass spectrometry (MS) were obtained from Millipore (MA, USA). All MS analyses were performed with a Voyager-DE STR MALDI-TOF instrument (Applied Biosystems, SCIEX, Toronto, Canada).
2.6 SDS-PAGE electrophoresis (RM, CM)
SDS polyacrylamide gel analyse of precipitated cell lysates was performed in order to separate protein-complexes. The samples were loaded onto a self-made, 4% stacking and 7,5% separation polyacrylamide gel using 1x running buffer. The gel was fixed in a 40% methanol solution, and stained with colloidal G-250 Coomassie blue.
2.7 In-gel trypsin/CNBr digestion (RM).
Peptide digestion of protein of interest to perform Mass Spectroscopy. Protein bands were cut from the gel and destained in 50% acetonitrile (ACN) until the gel pieces shrunk. ACN was removed and gel particles were dried by centrifugation under vacuum.
In-gel digestion was performed by adding 10 ng/μl trypsin in 50 mM ammonium hydrogen carbonate in quantities sufficient to moisten the gel sample spots. The well-hydrated samples were then incubated at 37°C overnight under stirring. After incubation the gel was separated from the hydrolysis solution and the supernatant was collected and saved. The gel spots were washed with 5% formic acid/50% ACN and further incubated for 10 minutes at RT. All the washes containing peptides were combined and dried in a vacuum concentrator. The supernatant was then harvested using a ZipTip micropipet and stored on ice.
2.8 MALDI/MS Analysis and data processing.
MALDI/MS analysis was performed in order to identify de binding-partners of Atg11p. Samples were applied to the MALDI-TOF plate and air dried. Subsequently, CHCA (10 mg/ml) in 50% acetonitrile, 0.1% TFA (v/v) was applied to the sample and dried again. MALDI mass spectra were recorded using a MALDI-TOF mass spectrometer using the reflectron mode. Ionization was performed with a 337 nm pulsed nitrogen laser. Spectra in the 750–4000 m/z mass range were accumulated and mass calibration was internally performed using molecular ions from matrix and trypsin autodigestion. Raw data were analyzed using Data Explorer software provided by the manufacturer and reported as monoisotopic masses.
The search engine Mascot peptide mass fingerprint (version 1.9.0; Matrix Science, London, UK) was used to process MS data, on a merged database SwissProt for in-house search, which contained the predicted proteins from all Fungi species. Non-redundancy of selected entries was carefully checked. Search parameters for MS spectra included: a maximum of zero incomplete tryptic cleavages allowed; peptide mass tolerance 0,3 Da; monoisotopic mass, 1+ peptide charge state as CHCA protonation, and oxidation as a variable modification.
3. Results
3.1 Fluorescence microscopy
Fluorescence analysis of GFP for localization analysis of Atg11 proteins and peroxisome proteins fused with GFP by using fluorescence microscopy.
Figure 5A: Fluorescence microscopy of SKL.GFP peroxisome degradation. Fluorescence microscopy of SKL.GFP cells expressing peroxisomal matrix marker GFP-SKL and were pre-grown at 37 °C on methanol medium, supplemented 0,5% glucose to induce peroxisome degradation.
Figure 5A shows the specific analysis of the peroxisome population present prior to the shift, by using peroxisomal matrix marker GFP-SKL under the control of the glucose-inducible alcohol oxidase promoter (PAOX).
Figure 5B: Fluorescence microscopy of Atg11p degradation-induced peroxisome degradation. Fluorescence microscopy of pdd18.GFP cells expressing GFP.Atg11p and were pre-grown at 37°C on methanol medium, supplemented 0,5% glucose to induce peroxisome degradation.
Figure 5B shows a microscopic image of macropexophagy induced Atg11p culture. This figure contains 1 strongly expressed GFP signal located in a vacuole (illustrated by number 1).
3.2 SDS PAGE
1D gel electrophorese analysis of Atg11p fused with GFP co-immunoprecipitated lysates for the identification of the interaction proteins of Atg11p involved in macropexophagy, by using SDS PAGE.
Figure 6: 1D Electrophoresis analysis of Atg 11p with binding partners. SDS-PAGE was carried out in a 5% stacking gel and 7,5% separation gel under denaturing conditions by β-ME from 4x SDS sample buffer and with boiling. Gels were visualized with Coomassie Brilliant Blue G-250 Dye. Proteins were purified with co-immunoprecipitation by using (2 µl serum/1 ml lysate) monoclonal mouse anti-GFP antibody ab1218 (Abcam). M = Precision Plus Protein™ Dual Color Standards marker (Bio-Rad). A: Lane 1 & 4: Supernatant Wash 1 from H. polymorpha Wild-Type NCYC495 strain; lane 2 & 5: Supernatant wash 2 from H. polymorpha Wild-Type NCYC495 strain; lane 3 & 6: Protein complex with Protein from H. polymorpha Wild-Type NCYC495 strain; lane 7: Supernatant Wash 1 from H. polymorpha Atg11.GFP; lane 8: Supernatant Wash 2 from H. polymorpha Atg11.GFP; lane 9: Supernatant Wash 1 from H. polymorpha Atg11.GFP.
B: Lane 1: Supernatant Wash 1 from H. polymorpha Atg11.GFP; lane 2: Supernatant Wash 2 from H. polymorpha Atg11.GFP; lane 3: Supernatant Wash 1 from H. polymorpha Atg11.GFP. Lane 4 & 7: Supernatant Wash 1 from H. polymorpha SKL.GFP NCYC495 strain; lane 5 & 8: Supernatant wash 2 from H. polymorpha SKL.GFP NCYC495 strain; lane 6 & 9: Protein complex with Protein from H. polymorpha SKL.GFP NCYC495 strain;
One-dimensional gel electrophorese analysis performed on two wash supernatants and one eluate from the glucose-induced macropexophagy cells and the not glucose-induced macropexophagy cells as control cells. The control cells in lane 4, 5, 6 in figure 6A and lane 1, 2, 3, 7, 8, 9 in figure 6B do not show protein bands. Precipitated glucose-induced macropexophagy Atg11p fused with GFP eluate in lane 9 figure 6A and precipitated glucose-induced macropexophagy SKL.GFP eluate in lane 4 in figure 6B contain a protein band of 74 kilo Daltons.
3.3 Mass Spectroscopy Analysis & Identification
Mass spectroscopy analysis to identify the interaction binding partners of Atg11p by using Peptide Mass Fingerprinting
Table 2: M/Z values and Peak intensity of unknown Protein found with co-IP
M/Z values peak intensity
1018 5258
1036 5748
1064 6076
1067 4879
1081 8938
1082 0985
1082 6017
1098 0163
1111 5404
1112 1989
1124 6486
1137 6481
1143 5017
1147 6326
1175 5036
1179 5406
1209 5744
1229 4671
1244 5871
1277 6393
1307 6666
1320 5536
1356 7172
1399 7013
MALDI-TOF m/z peak intensity sample gel 1 (fig. 6A) values was used for identification by MS fingerprinting because it gave a distinct pattern. The peak intensity and M/Z values are shown in table 2. Figure 7 was obtained by correlating the sample values with Mascot Search databases for MS fingerprinting. The database used a statistical approach to determine if the candidates are indeed likely to be in the sample. Parameters used for search: organism; fungi, peptide mass tolerance +/-0.1; significance threshold p< 0.05 gave a protein score -10*Log (P) of 72. The probability of the observed match found being a random event is a protein score below 58 sown in figure 7 by a green surface. The protein found was alcohol oxidase, further described in table 3. Top Score • 71 for ALOX_PICAN • Alcohol oxidase • OS=Pichia angusta • GN=MOX PE=1 SV=1 Table 3: Score protein identification Score Protein ID Protein Gene Organism 71 ALOX_PICAN Alcohol oxidase MOX Pichia angusta (= syn. Hansenula polymorpha)
4. Conclusion/ discussion
Conclusion
The aim of this research is to identify the binding-partner(s) of Atg11p that are involved in the metabolic pathway macropexophagy in the methylotrophic yeast Hansenula polymorpha. This suggests that Atg11p is an important component in peroxisome degradation and consistent with other studies that Atg11p recruits other proteins in the peroxisome-degradation. A similar peroxisome degradation process was observed in H. polymorpha cells with GFP tagged to Atg11p, upon exposure to methanol-excess conditions and during glucose induced macropexophagy. During this degradation process features of macropexophagy and peroxisome localization were observed. As for macropexophagy, the fluorescence microscopic image in figure 5A shows one expressed GFP signal, and for peroxisome localization, the fluorescence microscopic image in figure 5B shows a lot of GFP signals. Both results correspond to our expectation. Results from co-immunoprecipitation shows two protein bands of 74 kD; on gel 1 from sample glucose-induced macropexophagy Atg11p.GFP showed in figure 6A and on gel 2 from sample glucose-induced macropexophagy skl.GFP showed in figure 6B. Peptide Mass Fingerprinting reveals the identification of the found protein band from gel 1; Alcohol Oxidase from specie H. polymorpha.
Discussion
Alcohol oxidase can be cytosolic but is overall involved with methanol degradation in the peroxisomes, furthermore since alcohol oxidase functions only in the peroxisome, it is highly unlikely it is involved in the Atg11 protein complex. A more likely suggestion why alcohol oxidase was found is that during cell lyses the peroxisomes stayed intact and released alcohol oxidase during washing in co-immunoprecipitation. Alcohol oxidase is found in H. polymorpha samples strain SKl with glucose induction and PDD strain with glucose induction and thereby another reason that supports the suggestion of the Atg11 complex is not being associated with the alcohol oxidase. Although the chances of the alcohol oxidase being associated with the Atg11 protein complexes are small it is not excluded. Considering the possibility that the Atg 11 protein complex is involved further research should be stimulated. Final conclusion We found that optimization of the approach is needed to improve reproducibility and there by obtaining more convincing results. Convincing evidence of identification of Atg11p binding partners can be obtained by these methods. Although the approach of using co-immunoprecipitation to isolate the associated complex and using anti GFP is maybe not a very efficient way. Since the prevention of protein degradation is not easily done it could be efficient to optimize the buffer compounds during co-immunoprecipitation. So for further research, a more suitable method for protein purification could have been affinity chromatography, like a GST-tag cloned to Atg11p in H. polymorpha instead of a GFP tag or a tandem affinity purification.
References
1. Burnett, Sarah F. et al. “Peroxisomal Pex3 Activates Selective Autophagy of Peroxisomes via Interaction with the Pexophagy Receptor Atg30.” The Journal of Biological Chemistry 290.13 (2015): 8623–8631. PMC. Web. 14 June 2016.
2. Farré, Jean-Claude et al. “PpAtg30 Tags Peroxisomes for Turnover by Selective Autophagy.” Developmental cell 14.3 (2008): 365–376. PMC. Web. 14 June 2016.
3. Peroxisome homeostasis in Hansenula polymorpha, Adriana N Leão, Jan A.K.W. Kiel, FEMS Yeast Research Nov 2003, 4 (2) 131-139; DOI: 10.1016/S1567-1356(03)00070-9
4. Cox H, Mead D, Sudbery P, Eland M, Mannazzu I, Evans L. 2000. Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter. Yeast 16: 1191–1203.
5. Dijk R, Faber KN, Kiel JAWK, Veenhuis M, van der Klei I. 2000. The methylotrophic yeast Hansenula polymorpha: a versatile cell factory. Enzyme Microb Technol 26: 793–800.
Voor alle referenties geldt: bij meer dan 3 auteurs, alleen de naam van de eerste auteur, gevolgd door ‘e.a’. (= en anderen) Boeken: Auteurs, titel en ondertitel, druk, plaats, uitgever, jaar van uitgave, pagina nrs. Bijv. Smid O. en De Boer H., Rapporteren: een leidraad voor het schrijven van technische rapporten, 2e gewijzigde druk, Groningen, Wolters Noordhoff, 1993, blz. 104-119 Een hoofdstuk uit een boek: Auteurs, titel en ondertitel hoofdstuk, het woord ‘In’+ titel en ondertitel boek, druk, plaats, uitgever, jaar van uitgave, pagina nrs. Bijv. Jansen A. e.a., Het schrijven van medische verslagen. In: Smid O., en De Boer H. (eds) Rapporteren: een leidraad voor het schrijven van technische rapporten, 2e gewijzigde druk, Groningen, Wolters Noordhoff, 1993, blz. 104-119 Artikel in een tijdschrift: Auteurs, titel van het artikel, naam van het tijdschrift en editie/jaargang, jaar van uitgave, paginanummer Bijv. Redfern P.A., Neuromuscular transmission in newborn rats. J Physiol. 209; 1970; 709-711
Ongepubliceerd werk (scripties, rapporten, dictaten): Idem als voor boek, met aan het slot tussen haakjes het type werk, naam en afdeling van instelling of bedrijf Bijv. Jansen M. e.a., Puntlassen, Groningen 1992 (interne publicatie afdeling werktuigbouwkunde , Faculteit Techniek, Hanzehogeschool Groningen)
Internet: Zoveel mogelijk dezelfde manier van noteren Anderson, B. (2001), ‘Betrouwbaar onderzoeken’. In: De onderzoekers, jaargang 7, nr.4, Wolters Noordhoff: Groningen, (p.21-27). http://archief/nrc.nl/artikel bezocht (23-01-2002)
Appendix 1
FASTA >sp|Q67C55|ATG11_PICAN Autophagy-related protein 11 OS=Pichia angusta GN=ATG11 PE=3 SV=1
MNISIIQNNNPLSMMSSVYRQNQSQDTNLPSALTIYNSLTGAKVVASAYQFHNLEALKQF
IGMSFNVATENLFLLTPFGIKLKFSMIVHEEISEIYVFDRRYFNVNNIEASGNMDNVNDL
LAELNQTDFINMIKPLASPLLSEELSVFVEKLTLVLENTTQIKAVDINLNMLRMLLNSLK
RNSGWASALLSDFKKTVAFDECTPEDNDLETILTSLNVLIQYVGLVFKTLEKKFNDSIDA
LVLLQSNSLVDQWRDQYALLKRIPFEFKSGSSNVPEKLFLSQLVNESHLDKCAEESRRLN
KSMNERLVMLRSKIEADVIKPRQELLQEYNGYMSQYIRPETDATQKTQKIQDCKRILAEL
EVHVSKLIQSSSSLPSFEELITTASQTSTTLSASSIANIKKLTQLYKYQESELVPYIFQL
ANNLYDIQINKLNARKELQTKLICSTLINITKIQLNIMRLSTVLNTEVAKNIASIKENEL
QLSVVSDLPLMFGIFVIANLNNLKFGISLNNIVKKANEIFEMLRFMESRNRAKWLKEFLA
SSGADKVEFLHLDEEARERFINENMLSYKLEQVDAIRSKKSPSPVDHPASPVSGQEKHYL
TSINRLLHNINGFPAPRPTATELPKQRETNIMTNLARNISVKSIVSYINTLRKEGIDLNI
VNRLEECLKDFGITYGAIERKAIETEDGEEIVVKGKNAGDLGTFDVNDVNYMRLFKKFIK
SFESEGIVININVNQQDSVSNDELIKGYERRIRKLENVLHTRNFQQFNEQWSRHRPVHTL
PNPVSRRQSDMSQEPAVHENTILFNENVVLGRKTIDLPPSHYGERIERLEKENERYRGEI
EELKKGTDLAELDRLKKEIEDLQKADMEKDKRLAALEEENKNLKESNEELTNSNKELVNM
CEELKSMKSDLLENMTQKESEFGKEAKVNQQEINELKLRIEELEEDESNLVNVNKTLNER
LAIKDGLLCQLYELVQGAYGKLNQMSGEIFSNLTRVCLLLESIGLLLIRETPSFDNHPGT
LTIKRVKGLRSRKRQIKQASDSTHNGNLQNDTFEDSEHIDNALMEVVSSEVVPEAEQYLH
WVDTNVLNYTISSDLGIEDEIEHKKNESSLIDMSLCEESSIEKKVKKLLANYESFNVEQG
FQNFLRFNHVDNELVIERVFRRFSDVETLARKLQKDKTQQKQELKMLTAELDGKIAFRNF
KVGDLVLFLKTLTPANEELGGGDEQPWAAFNVGCPNYYLKNTKGEGYIELSDRDWLVGRV
SKIEPRQVTEQNFHSKTENPFRLAKSVVWYYVEAREVKE
Appendix 2
Table 4
Sample Concentratie OD OD-STB
A 2000 1,676 1,438
B 1500 1,339 1,101
C 1000 1,067 0,829
D 750 0,868 0,63
E 500 0,58 0,342
F 250 0,366 0,128
G 125 0,315 0,077
H 25 0,244 0,006
Table 5
Sample Naam OD OD-SB Concentration
1 Wt- 0,433 0,263 381,2857143
2 Wt+ 0,408 0,238 345,5714286
3 pdd- 0,237 0,067 101,2857143
4 pdd+ 0,315 0,145 212,7142857
5 skl- 0,347 0,177 258,4285714
6 skl+ 0,457 0,287 415,5714286
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