Supporting information
S1
S1: Heterologous NLSs used in this work. The NLSs were selected, because they are most commonly used in literature for
the import of heterologous proteins. Heterologous sequences which showed NLS function in P. pastoris mentioned in the main
manuscript (the bovine gamma interferon NLS (Gradoboeva & Padkina, 2010), the NLS of the human topoisomerase I (Yang
et al., 2004)) were not tested, since they have never been used independently from the native protein context.
Abbreviatio
Protein name
Source
NLS - Protein sequence
SV40 large T
Simian Virus 40
PKKKRKV
CCAAAGAAGAAAAGAAAAGTT
n
SV40
DNA sequence codon optimized for
References
P. pastoris
(Lanford
&
Butel, 1984)
antigen
ScMatα2
Matα2
S. cerevisiae
KIPIK
AAGATTCCAATTAAG
HsMyc
c-Myc
H. sapiens
PAAKRVKLD
CCAGCTGCTAAGAGAGTTAA
(Dang & Lee,
GTTGGAT
1988)
XlNuc
Nucleoplasmin
X. leavis
KRPAAATKKAGQAK
AAGAGACCTGCTGCTGCCAC
(Conti
KKK
TAAGAAAGCAGGGCAAGCTA
Kuriyan,
AGAAGAAGAA G
2000)
KYENVVIKRSPRKRG
AAGAAGTACGAAAACGTTGTT
(Jans, 1995)
RPRK
ATCAAGAGATCCCCAAGAAAG
(Hall et al.,
1984)
ScSWI5
SWI5
S. cerevisiae
AGAGG UAGACCAAGAAAA
1/12
&
S2
S2: P. pastoris endogenous NLSs. NLSs of putative nuclear proteins were selected in the genome sequence of P. pastoris (De
Schutter et al., 2009), according to the published consensus motifs (Chook & Blobel, 2001). The P. pastoris (Pp) proteins were
used as queries for a BLAST search in S. cerevisiae, demonstrating that they are homologs of the S. cerevisiae nuclear proteins.
Information on the function of the homologous proteins from S. cerevisiae taken from the Saccharomyces Genome Database
(Cherry et al., 2012) is provided.
Abbreviation
PpNob1
Genbank
S.
Putative nuclear import
Homologous protein function
accession
cerevisiae
motif
in S. cerevisiae
number
homolog
XM_002493229
Nob1p
KGRRANASKKKK
p-Blast results
Protein involved in proteasomal
Query coverage:
and
98%
40S
ribosomal
subunit
biogenesis; required for cleavage
of the 20S pre-rRNA to generate
the mature 18S rRNA;
PpSda1
XM_002490388
Sda1p
KQKVLRAHIDKQKKKGH
Protein
required
E value: 2e-114
Identity: 42%
for
actin
organization and passage through
Query coverage:
100%
Start; highly conserved nuclear
protein;
required
for
actin
cytoskeleton organization; plays
E value: 0.0
Identity: 55%
a critical role in G1 events;
PpSet7
XM_002491385
Set7p
KRKLEEEEGSKRNKRIKG
Ribosomal
lysine
methyltransferase; specific for
Query coverage:
98%
monomethylation of Rpl42ap
and
Rpl42bp
(lysine
55);
Location nucleus
PpUba1
XM_002490958
Uba1p
KRPLEIEQEETYSKRKKSTI
E value: 3e-96
Identity: 39%
Subunit of heterodimeric nuclear
SUMO activating enzyme E1
Query coverage:
32%
with Aos1p; activates Smt3p
E value: 1e-62
(SUMO) before its conjugation
to proteins (sumoylation), which
Identity: 61%
may play a role in protein
targeting; essential for viability
PpSwi5
XP_002489440
Swi5p
KKFVRNHDLRRHKKK
Transcription factor that recruits
Mediator
and
complexes;
Swi/Snf
Query coverage:
21%
activates
E value: 1e-33
transcription of genes expressed
at the M/G1 phase boundary and
in
G1
phase;
required
for
expression of the HO gene
controlling
mating
type
switching; localization to nucleus
occurs during G1 and appears to
be regulated by phosphorylation
by Cdc28p kinase;
2/12
Identity: 52%
S3: Full materials and methods.
A – P. pastoris strain generation
The plasmids used for the expression of the eGFP-NLS fusion constructs are based on the P.
pastoris pPpT4_S vector (Näätsaari et al., 2012) and the eGFP reporter gene sequence
previously reported for promoter comparisons (Vogl et al., 2014). The NLSs were seamlessly
fused to the eGFP CDS sequence without a linker sequence. The heterologous NLSs have been
codon optimized for the expression in P. pastoris. The DNA sequences of the NLSs were either
fused to eGFP with primers or by the aid of synthetic double stranded DNA fragments
(gBlocks) (Table SA1, SA2). The plasmids were assembled by Gibson assembly (Gibson et al.,
2009) and the insert sequence and its flanking regions were verified by Sanger sequencing. All
plasmids had been SwaI-linearized and transformed into the P. pastoris CBS7435 wildtype
strain using a condensed electroporation protocol (Lin-Cereghino et al., 2005).
Table SA1
Primer name
Sequence 5' --> 3'
pAOX1-nNLS SV40-eGFP-Gib
PGAP-nNLS-XlNuc-fw
GACAActtgagaagatcaaaaaacaactaattattcgaaacgATGCCAAAGAAGAAAAGAAAAGTTGC
TAGCAAAGGAGAAGAACTTTTCACTG
GACAActtgagaagatcaaaaaacaactaattattcgaaacgATGCCAGCTGCTAAGAGAGTTAAGTT
GGATGCTAGCAAAGGAGAAGAACTTTTCACTG
GACAActtgagaagatcaaaaaacaactaattattcgaaacgATGAAGAGACCTGCTGCTGCCACTAA
GAAAGCAGGGCAAGCTAAGAAGAAGAAGGCTAGCAAAGGAGAAGAACTTTTCA
CTG
GACAActtgagaagatcaaaaaacaactaattattcgaaacgATGAAGATTCCAATTAAGGCTAGCAA
AGGAGAAGAACTTTTCACTG
GCAAATGGCATTCTGACATCCTCTTGATTAAACTTTTCTTTTCTTCTTTGGCTTGT
ACAATTCATCCATGCCATGTGT
GCAAATGGCATTCTGACATCCTCTTGATTAATCCAACTTAACTCTCTTAGCAGCT
GGCTTGTACAATTCATCCATGCCATGTGT
GCAAATGGCATTCTGACATCCTCTTGATTACTTCTTCTTCTTAGCTTGCCCTGCTT
TCTTAGTGGCAGCAGCAGGTCTCTTCTTGTACAATTCATCCATGCCATGTGT
GCAAATGGCATTCTGACATCCTCTTGATTACTTAATTGGAATCTTCTTGTACAAT
TCATCCATGCCATGTGT
GACAActtgagaagatcaaaaaacaactaattattcgaaacgATGGCTAGCAAAGGAGAAGAACTTTT
CACTG
GCAAATGGCATTCTGACATCCTCTTGATTACTTGTACAATTCATCCATGCCATGT
GT
TATTTCAATCAATTGAACAACTATCAAAACACAATGGCTAGCAAAGGAGAAGAA
CTTTTC
TATTTCAATCAATTGAACAACTATCAAAACACAATGAAGAGACCTGCTGCTGC
pUC-PTPI-fw
CTGAAAAATACACAGTTATTATTCATTTAAATTCAACGAGACACTCTTCCGTCAG
GFP-PTPI-rv
nNLS-XlNuc-PTPI-rv
GAAAAGTTCTTCTCCTTTGCTAGCCATTGTGTTTGTGATAGATCTTGTATATCAAT
G
GCAGCAGCAGGTCTCTTCATTGTGTTTGTGATAGATCTTGTATATCAATG
pTPI-nNLS-XlNuc-fw
ATGAAGAGACCTGCTGCTGC
pGAP-sTOM-fw-Gib
CCCTATTTCAATCAATTGAACAACTATCAAAACACAATGGTTTCTAAGGGTGAG
GAAGTTATCAAG
CAAATGGCATTCTGACATCCTCTTGATTACTTATAAAGCTCGTCCATACCGTACA
AGAA
pAOX1-nNLS c-myc-eGFP-Gib
pAOX1-nNLS nucleoplasmin-eGFP-Gib
pAOX1-nNLS tr Matα2-eGFP-Gib
AOX1TT-cNLS SV40-eGFP-Gib
AOX1TT-cNLS c-myc-eGFP-Gib
AOX1TT-cNLS nucleoplasmin-eGFPGib
AOX1TT-cNLS tr Matα2-eGFP-Gib
pAOX1-eGFP-Gib
AOX1TT-eGFP-Gib
PGAP-GFP-fw
AOXTT-sTom-rv-Gib
3/12
Table SA2
gBlock-cNLS-PpNob1
CCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACG
AAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGAT
GAATTGTACAAGAAAGGAAGGCGGGCTAATGCCTCAAAGAAGAAGAAGTAATCAAGAGGATGT
CAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATA
GTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGCCTATCTCGCA
GCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTTTCTTGGTATT
TCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCGGATCCTTCAGTAAT
GTCTTGTTTCTTTTGT
gBlock-cNLS-PpSda1
CCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACG
AAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGAT
GAATTGTACAAGAAGCAAAAGGTTCTACGAGCTCATATCGATAAGCAAAAAAAGAAGGGTCAT
TAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTT
ATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTG
ATCAGCCTATCTCGCAGCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGA
TGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGC
GGATCCTTCAGTAATGTCTTGTTTCTTTTGT
gBlock-cNLS-PpSet7
CCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACG
AAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGAT
GAATTGTACAAGAAGAGAAAACTTGAAGAAGAGGAAGGGTCAAAGAGAAACAAACGGATAAA
AGGTTAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTT
TTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCT
CCTGATCAGCCTATCTCGCAGCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGT
TTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTT
GTGCGGATCCTTCAGTAATGTCTTGTTTCTTTTGT
gBlock-cNLS-PpUba1
CCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACG
AAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGAT
GAATTGTACAAGAAGCGACCTCTGGAAATAGAGCAGGAAGAAACATATTCGAAAAGAAAGAAG
AGTACTATATAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGA
TACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGC
TTGCTCCTGATCAGCCTATCTCGCAGCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATT
CGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTT
CGTTTGTGCGGATCCTTCAGTAATGTCTTGTTTCTTTTGT
gBlock-cNLS-PpSwi5
CCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACG
AAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGAT
GAATTGTACAAGAAAAAGTTCGTCAGAAATCATGATCTTCGAAGGCATAAAAAGAAATAATCAA
GAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTATTTGTA
ACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCCTGATCAGC
CTATCTCGCAGCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGAGTTTGATGTTTT
TCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCTTCGTTTGTGCGGATC
CTTCAGTAATGTCTTGTTTCTTTTGT
gBlock-cNLS-ScSwi5
CCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACG
AAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGAT
GAATTGTACAAGAAGAAGTACGAAAACGTTGTTATCAAGAGATCCCCAAGAAAGAGAGGUAGA
CCAAGAAAATAATCAAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTG
ATACTTTTTTATTTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAG
CTTGCTCCTGATCAGCCTATCTCGCAGCAGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCAT
TCGAGTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGAGACCT
TCGTTTGTGCGGATCCTTCAGTAATGTCTTGTTTCTTTTGT
4/12
B – P. pastoris cultivation protocols
The transformants were cultivated in 96-deep well plates (Bel-Art Products, USA) for 60 hours
and methanol induction was performed for 24 hours (Weis et al., 2004). Roughly 84
transformants per construct were screened for eGFP expression and subsequently four
representative transformants from the landscape were rescreened. Cell suspensions (1:20
dilution) from the 96-deep well plates were added to a clear bottom fluorescence plate (Thermo
Scientific, USA) and the optical density at 600 nm and eGFP fluorescence were determined
using a SynergyMX platereader (BioTek, USA) with 488 nm excitation and 507 nm emission
wavelength. One representative transformant of four rescreening transformants of each
construct was selected for the microscopy experiments in order to investigate eGFP transport.
The fluorescence of the cells under the microscope was very intense and it was not possible to
localize single cellular compartments after the cultivation on methanol for 24 hours, because of
the high levels of intracellular eGFP (Figure SB1, 24 h MeOH). The eGFP expression was
repressed by the addition of glucose to the cultivation media and a decrease in induction time
(Figure SB1, 4 h MeOH, 4 h glucose). All strains, observed under the microscope, were
cultivated accordingly: The cells were grown for 60 hours in BMD1 and induced with 250 µl
BMM2 and grown for four hours (Weis et al., 2004). Subsequently 0.5% glucose (50 µl
BMD10) was added and cultivation was prolonged for additional four hours. The strains were
then either directly used for the microscopy experiments or stored at 4°C overnight. If the
repression step with glucose is omitted, less eGFP is transported to the nucleus and the
cytoplasmic background signal is brighter (Figure SB1, 8 h methanol). Figure SB2 shows the
relative fluorescence levels measured with a spectrophotometer after 24 hours growth on
methanol and after 4 hours methanol induction and additional 4 hours growth on glucose. Less
eGFP is produced, when the induction time is reduced.
Notably, already after 60 h growth on glucose (BMD1 media) very weak eGFP fluorescence is
visible. In the construct with the NLS, even nuclear targeting seems weakly apparent. We
assume this is effect is due to derepression of the AOX1 promoter used for expression: PAOX1 is
completely repressed on carbon sources such as glucose. Once the glucose in the media is
depleted, PAOX1 shows weak derepression to about 2-4 % of methanol induced levels (Vogl &
Glieder, 2013). In our experimental setup, cells were grown past glucose depleteion reaching
the derepressed state. Apparently the weak derepressed expression from PAOX1 is sufficient to
generate enough protein resulting in dedectable fluorescence levels for microscopy.
5/12
Figure SB1: eGFP fluorescence obtained with different cultivation protocols. Fluorescence microscopy images of the P.
pastoris CBS 7435 strains expressing GFP fused to a representative functional NLS (PpSet7 C-terminally fused to eGFP),
eGFP without a NLS (w/o eGFP) and the wildtype strain during the time course of different cultivation conditions. All cells
were inoculated in a 96-DWP containing 250 µl BMD1 media and grown for 60 h (t0). After 60 h an induction step with
methanol was performed and the cells were grown for four hours (t1). Then the cells were either induced with glucose
containing media (50 µl BMD10, G) or kept shaking, omitting the induction step (M). After 4 hours cultivation fluorescence
images of the differently cultivated cells were taken (t2M, t2G). The cells, which had been solely induced with methanol, were
repeatedly induced with methanol (50 µl BMM10) after 12 hours. Twenty-four hours after the first methanol induction step
images of cells, which were grown on methanol (24 h MeOH) and of cells, which were grown on methanol and glucose, were
taken.
6/12
Relative fluorescence units / OD600 [%]
Figure SB2: Quantitative fluorescence spectroscopy measurements of eGFP-NLS fusion constructs using different
cultivation protocols. The relative fluorescence levels of the eGFP-NLS fusion constructs to the control (GFP localized in the
cytoplasm, after 24 hours growth on methanol) are shown. The fluorescence levels of the strains expressing various NLS-GFP
fusion proteins were measured after 24 hours growth on methanol and after four hours growth on methanol plus additional four
hours growth on glucose. The CBS 7435 wildtype (WT) strain does not express eGFP. Mean values and standard deviations of
biological 6-fold replicates are shown.
120
24 h growth on methanol
100
4h growth on methanol +
4h growth on glucose
80
60
40
20
0
C – P. pastoris nuclear staining protocol
The morphology of the cells and the translocation of eGFP were observed with a Leica DM LB
bright field and fluorescence microscope (Leica Mikrosysteme GmbH., Austria). The nuclear
localization of eGFP was confirmed by Hoechst 33258 (Sigma-Aldrich GmbH, Austria)
staining. Five hundred µl cell culture were removed from the 96-DWP and transferred into an
Eppendorf tube (Eppendorf, Hamburg, Germany). Five hundred µl PBS buffer, pH 7.4, 25 µl 1
M DTT and 5 µg Hoechst 33258 were added and the cells were stained for two hours at 28°C,
at 1000 rpm shaking. For the nuclear stain images a microscope filter with 355-425 nm
excitation wavelengths and 470 nm suppression wavelength was used. The eGFP-fluorescence
was observed with a filter of excitation wavelengths between 450 and 490 nm and 515 nm
suppression wavelength.
7/12
S4
S 4: Expression from constitutive promoters (PGAP and PTPI) results in identical nuclear targeting
as from the methanol inducible AOX1 promoter (Fig. 1).
8/12
Caption
(a) Quantitative fluorescence spectroscopy measurements of eGFP-NLS fusion constructs using the
constitutive GAP and TPI promoters. The relative fluorescence levels of the eGFP-NLS fusion
constructs relative to the control (eGFP localized in the cytoplasm) after 60 hours growth in YPD or
BMD1 media are shown. The CBS 7435 wildtype (WT) strain does not express eGFP. Mean values and
standard deviations of biological six-fold replicates are shown.
(b, c) Fluorescence microscope images of eGFP-NLS fusion constructs measured spectroscopically in
panel A. The XlNuc was fused N- and C-terminally (indicated by prefixes N- and C-) to an eGFP reporter
gene and transformed in P. pastoris. The reporter gene was expressed from either PGAP or PTPI. The
nuclei were stained with Hoechst 33258. Bright field images (BF) and fluorescence images using
different filters are shown (Hoechst, eGFP). As a localization control the eGFP gene was expressed
without a NLS (w/o NLS).
Experimental outline and discussion of results
The AOX1 promoter is the most commonly used promoter for protein expression in P. pastoris (Vogl &
Glieder, 2013). However, methanol induction required to activate the promoter also causes an increase
in the size of peroxisomes (van der Klei et al., 2006) and might influence nuclear targeting. Thus we
also expressed NLS fusion constructs under two constitutive promoters. The XlNuc translocation
sequence was fused either N- or C-terminally to the eGFP CDS and cloned into plasmids based on
pPpT4_S (Näätsaari et al., 2012) bearing either the strong constitutive promoter of the glyceraldehyde
3-phosphate dehydrogenase (GAP) gene or the weak constitutive promoter of the triosephosphate
isomerase (TPI) gene (Vogl & Glieder, 2013) (primers: Table SA1).
The plasmids were transformed in the P. pastoris CBS 7435 wildtype strain and transformants were
cultivated in BMD1 media (minimal media) and YDP media (rich media) for 60 hours in 96-DWPs.
Figure S 4A shows the relative fluorescence levels measured with a spectrophotometer after 60 hours.
Using YPD media more than 2-fold higher fluorescence levels were obtained compared to cells grown
in BMD1 media (Figure S 4A).
It was possible to observe nuclear translocation under the microscope with the XlNuc fusion constructs
independently from the cultivation media or promoter used (Figure S 4B,C). However the cultivation in
YPD media resulted in high eGFP expression and strong cytoplasmic background signals with both the
weak TPI promoter and the strong GAP promoter. Expression by PGAP appears too strong, filling the
whole cytosol and likely overburdening the nuclear import machinery. This effect was also visible for
the strong AOX1 promoter, where we quenched expression by glucose addition (Figure SB1).
Fluorescence images similar to Figure 1 (PAOX1, 4h methanol induction + 4h glucose repression) were
obtained using the weak constitutive TPI promoter and BMD1 media for the cultivation. In general YPD
media was less suitable for microscopy experiments since auto-fluorescence had been observed and
washing of the cells (with 1xPBS) was required, the nuclear dye Hoechst 33258 was less soluble and
high eGFP fluorescence levels were obtained, which hampered a clear distinction between active and
inactive targeting sequences.
9/12
S5
S5: Summary table on functions and conclusions of the NLSs in P. pastoris. The table summarizes the performance of
endogenous and heterologous NLS fused to eGFP. The translocation depends on the protein context and we recommend testing
several NLSs, when trying to target a protein of interest to the nucleus. NLSs, which are functional, when fused to eGFP, might
not work with a protein displaying a different structural organization and vice versa.
NLS
eGFP fusion protein
Relative eGFP expression levels
compared to the control (w/o NLS)
[%]
Nuclear
translocalization
observed under
microscope
SV40
PpNob1
n-terminal
c-terminal
n-terminal
c-terminal
n-terminal
c-terminal
n-terminal
c-terminal
n-terminal
c-terminal
c-terminal
20.8±1.3
61.2±2.9
78.0±13.3
73.0±5.3
71.1±2.7
87.0±6.6
74.9±7.0
86.2±8.8
59.3±8.0
62.6±15.0
70.1±4.1
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
PpSda1
c-terminal
72.7±4.9
No
PpSet7
c-terminal
27±2.1
Yes
PpUba1
c-terminal
29.4±2.3
Yes
PpSwi5
c-terminal
57.3±6.5
No
HsMyc
XlNuc
ScMatα2
ScSWI5
10/12
S6
S6: mCherry and dTomato show mistargeting in P. pastoris, complicating its application as
reporter for evaluating intracellular localization of fusion proteins/peptides in P. pastoris. The
untagged reporter genes were expressed from PGAP. Bright field images (BF) and fluorescence images
using the respective filters are shown (FluMi).
Experimental outline and discussion of results
We also intended to test other fluorescence reporter proteins additionally to eGFP for the
characterization of the NLSs. We selected two red fluorescent protein (RFP) variants mCherry
and dTomato (Shaner et al., 2004). Previous to nuclear targeting experiments, we performed
microscopy studies of the untagged reporter proteins as negative controls to rule out
interference with cellular localization. We also included eGFP as control.
All reporter genes were expressed from the strong constitutive GAP promoter in P. pastoris
CBS 7435 wildtype cells. The construction of the eGFP expressing strain was described in S3
and S4. The dTomato gene sequence (previously reported (Geier et al., 2015)) was cloned in a
plasmid based on pPpT4_S (Näätsaari et al., 2012) bearing the strong constitutive GAP
promoter with the primers GAP-sTom-fw and AOXTT-sTom-rv listed in Table SA1. A P.
pastoris strain expressing mCherry was gathered from the strain collection (#3179) of the
Institute for Molecular Biotechnology, Graz University of Technology, Petersgasse 14/2, 8010
Graz, Austria. The construction of this strain is described in the Master’s thesis of Manuel Peter
“Evolution of fluorescent reporter proteins for the gene expression analysis in Pichia pastoris”
(publication date December 2007). Cell material of the P. pastoris strains from YPD-Zeocin
(100µg/ml) plates was resuspended in 200 µl 1xPBS and used for fluorescence microscopy
(Figure S6).
It appears that mCherry as well as dTomato are unsuitable for targeting experiments: In contrast
to eGFP, which shows uniform cytosolic fluorescence, the untagged mCherry and dTomato
proteins are mistargeted to a large compartment, presumably the vacuole. Apparently mCherry
and dTomato contain a cryptic targeting signal recognized by P. pastoris, troubling its use as
reporter for cellular localization studies in P. pastoris.
11/12
Supporting references
The supporting references do not appear in the main manuscript text and the main reference
list.
Geier M, Fauland P, Vogl T & Glieder A (2015) Compact multi-enzyme pathways in P. pastoris. Chem
Commun 51: 1643–1646.
Gibson DG, Young L, Chuang R, Venter JC, Hutchison CA & Smith HO (2009) Enzymatic assembly
of DNA molecules up to several hundred kilobases. Nat Methods 6: 343–345.
Lin-Cereghino J, Wong WW, Xiong S, Giang W, Luong LT, Vu J, Johnson SD & Lin-Cereghino GP
(2005) Condensed protocol for competent cell preparation and transformation of the
methylotrophic yeast Pichia pastoris. Biotechniques 38: 44, 46, 48.
Näätsaari L, Mistlberger B, Ruth C, Hajek T, Hartner FS & Glieder A (2012) Deletion of the Pichia
pastoris KU70 homologue facilitates platform strain generation for gene expression and synthetic
biology. PLoS One 7: e39720.
Van der Klei IJ, Yurimoto H, Sakai Y & Veenhuis M (2006) The significance of peroxisomes in
methanol metabolism in methylotrophic yeast. Biochim Biophys Acta - Mol Cell Res 1763: 1453–
1462.
Vogl T, Ruth C, Pitzer J, Kickenweiz T & Glieder A (2014) Synthetic Core Promoters for Pichia
pastoris. ACS Synth Biol 3: 188–191.
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screening with Pichia pastoris by limiting yeast cell death phenomena. FEMS Yeast Res 5: 179–
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