Gene specific primers. Left border primer (638) ala3-4 (Salk_082157) Gene specific primers. Left border primer (1343)

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Supplemental Data. Poulsen et al. (2008) The Arabidopsis P4-ATPase pump ALA3 localizes to the Golgi and requires a β subunit to function in lipid translocation and secretory vesicle formation.

1 Supplemental Data. Poulsen et al. (2008) The Arabidopsis P4-ATPase pump ALA3 localizes to the Golgi and requires a β subunit to function in lipid translocation and secretory vesicle formation. A ala3-1 (SAIL_422_C12) WT Gene specific primers Left border primer (638) B ala3-4 (Salk_082157) WT Gene specific primers Left border primer (1343) Supplemental Figure 1. PCR analysis of ala3 mutant lines. No gene specific PCR product for ALA3 could be amplified in ala3-1 (A) or ala3-4 (B) mutant plants. The inserted T-DNA was detected in these plants using a left border primer in combination with an ALA3 gene specific primer. Primers used are listed in Supplemental Table 1.

Supplemental Figure 2. A B C D E F Supplemental Figure 2.

A fusion protein of GFP:ALA3 was transiently expressed in tobacco

A Leica TCS SP2/MP confocal laser scanning microscope with a 63x/1.

GFP was excited at 488 nm and emission was recovered in the interval

YFP was excited at 514 nm and fluorescent emission measured at 525-540

2 Supplemental Figure 2. A B C D E F Supplemental Figure 2. Movement of intracellular bodies containing ALA3. A fusion protein of GFP:ALA3 was transiently expressed in tobacco epidermal cells in concert with a Golgi marker ST:YFP. A Leica TCS SP2/MP confocal laser scanning microscope with a 63x/1.2 NA water immersion objective was used. GFP was excited at 488 nm and emission was recovered in the interval nm. YFP was excited at 514 nm and fluorescent emission measured at nm. Sequential scanning between lines was used to follow both fluorescent proteins at once. Overlay images of YFP and GFP are shown. Arrows mark positions of intracellular Golgi bodies. Images are taken with sec intervals from A to F.

30 C 18 C GFP tagged versions of ALA3 and ALIS1 are functional.

of a galactose inducible promoter and found to functionally complement the cold sensitive

Serial 10-fold dilutions of cells were spotted onto medium containing glucose (GLU) or

3 Supplemental Figure 3. Empty Glu Gal Gal Glu Gal drs2 dnf1 dnf2 GFP:ALA3 GFP:ALA3/ALIS1 GFP:ALIS1 ALA3/GFP:ALIS1 30 C 18 C Supplemental Figure 3. GFP tagged versions of ALA3 and ALIS1 are functional. GFP:ALA3 was expressed with ALIS1 in the triple yeast mutant drs2 dnf1 dnf2 under control of a galactose inducible promoter and found to functionally complement the cold sensitive phenotype of this strain. The same is the case for GFP:ALIS1 when expressed with ALA3. Serial 10-fold dilutions of cells were spotted onto medium containing glucose (GLU) or galactose (GAL). Plates were scanned after 2-3 d of incubation at 30 C or after 6-8 d at 18 C, as indicated. Empty, yeast cells transformed with a control plasmid.

Supplemental Figure 4. A B C D E Supplemental Figure 4.

A fusion protein of GFP:ALIS1 was transiently expressed in tobacco

4 Supplemental Figure 4. A B C D E Supplemental Figure 4. Movement of intracellular bodies containing ALIS1. A fusion protein of GFP:ALIS1 was transiently expressed in tobacco epidermal cells in concert with a Golgi marker ST:YFP. A Leica TCS SP2/MP confocal laser scanning microscope with a 63x/1.2 NA water immersion objective was used. GFP was excited at 488 nm and emission was recovered in the interval nm. YFP was excited at 514 nm and fluorescent emission measured at nm. Sequential scanning between lines was used to follow both fluorescent proteins at once. Overlay images of YFP and GFP are shown. Arrows mark positions of intracellular Golgi bodies. Images are taken with sec intervals from A to E.

Supplemental Figure 5. A B C D E F Supplemental Figure 5.

conditions: A Leica TCS SP2/MP confocal laser scanning microscope with a 63x/1.

(A to C) Tobacco epidermal cells expressing a GFP:ALIS1 fusion.

(A) bright field (B) Detection of YFP signal, emission recovery in the interval

495-512 nm. (D to F) Tobacco epidermal cells expressing the Golgi marker ST:YFP.

5 Supplemental Figure 5. A B C D E F Supplemental Figure 5. Bleed-through analysis of GFP and YFP fluorescence under routinely experimental conditions: A Leica TCS SP2/MP confocal laser scanning microscope with a 63x/1.2 NA water immersion objective was used. (A to C) Tobacco epidermal cells expressing a GFP:ALIS1 fusion. Excitation wavelength for GFP was 488 nm. (A) bright field (B) Detection of YFP signal, emission recovery in the interval nm (C) Detection of GFP fluorescence, emission recovered in the interval nm. (D to F) Tobacco epidermal cells expressing the Golgi marker ST:YFP. Excitation wavelength for YFP was 514 nm. (D) bright field (E) Detection of YFP signal, emission recovery in the interval nm (F) Detection of GFP fluorescence, emission recovered in the interval nm.

6 Supplemental Figure 6. Fraction number Pma1p Dpm1p Sed5p HA-ALA3 RGSH6-ALIS1 Supplemental Figure 6. Small amounts of ALA3 and ALIS1 can be detected in the yeast plasma membrane. Yeast expressing ALA3 and ALIS1 were lysed, subjected to differential centrifugation (500 x g for 10 min; 9,000 x g for 20 min), and membrane pellets from the 9,000 x g centrifugation enriched in the plasma membrane were loaded on top of a step gradient consisting of 53% and 43% (w/w) sucrose in lyses buffer. Gradient fractions were assayed by immunodetection using antibodies against the HA epitope, RGSH 6 epitope or several organelle markers. The ER marker Dpm1p can only be observed in fractions number 1 and 2, while the Golgi-localized protein Sed5p is mainly present in fractions 1-3. This Golgi protein can also be detected in small amounts in fraction 6 and 7, where the highest signal for the plasma membrane marker Pma1p is found. ALA3 and ALIS1 follow the same localization pattern and can be detected in the Golgi fractions, but also in those corresponding to the plasma membrane.

7 Supplemental Table 1. Primers used for knock out identification Knock out line Primer name Sequence ala3-1 SAIL_422_C a 1610br 5'-GAGATAGATATAGATAGAGACCCACTT-3' 5'-AGACAAACATCTAAACAAATGATGGAA-3' 638 5'-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3' ala3-4 Salk_ a 1629br 5'-TCTTCGGCGACTCATCAACGGACTCCGTCTCGAA-3' 5'-GAGACTCCAACGCATCTACTAACTTGCACAGATT-3' '-CTCGGGCTATTCTTTTGATTTATAAGGGATTTTGC-3' Seed stock numbers for plant lines used in this work Name Gene Description ss834 ala3-1 SAIL_422_C12; Sessions et al., 2002 ss836 ala3-4 Salk_082157; Alonso et al., 2003 ss ala3-1 /GFP:ALA3 ala3-1 transformed with ps1019; hyg r smp75 WT /ProALA3:GUS Wild type transformed with pmp2347; hyg r smp76 WT /ProALIS1:GUS Wild type transformed with pmp2747; hyg r

8 Supplemental Table 2. Primers used for RT-PCR Target gene Primer name Sequence ALIS1 Oli_2533B 5'-CCAAATAAACCA^TTGAG-3' ALIS2 Oli_2534B 5'-GGATCCAAAAATTCCA^CTAAG-3' ALIS3 Oli_2535B 5'-CCAAGAATACCG^CTTAG-3' ALIS4 Oli_2536B Oli_1951 5'-CAAGATATTCCA^TTGAG-3' 5'-TTAGAGCTCCTCGAGTCATCTTCCACCTCCAGCAC-3' ALIS5 Oli_2537B 5'-CTTAATATTTCTAAACCG^TTGAG-3' ACTIN3 Oli_896 Oli_897 5'-TGGAACTGGAATGGTTAAGGCTGG-3' 5'-TCTCCAGAGTCGAGCACAATACCG-3' ^ Intron position. Reverse primers of ALIS1, ALIS2, ALIS3 and ALIS5; Oli_1945, Oli_1947, Oli_1949 and Oli_1953; see Supplemental Table 3.

9 Supplemental Table 3. Primers used for cloning Target gene Primer name Sequence ALA3 Oli_1995 Oli_1997 Oli_1996 5'-CACCGAATTCATGGTTCGATCGGGTAGTTTTAG-3' 5'-TTACTTCTTCGGTACCTTTGGTCTAGATCTCATAC-3' 5'-CTTCTTCGGTACCTTTGGTCTAGATCTCATAC-3' HA-ALA3 Oli_2089 5'-CCGGAATTCATGTACCCATACGATGTTCC AGATTACGCTGTCGATCGGGTAGTTTTAGCG-3' ala3d413a ALIS1 RGSH 6 - ALIS1 ALIS2 RGSH 6 - ALIS2 ALIS3 RGSH 6 - ALIS3 ALIS5 RGSH 6 - ALIS5 Oli_2321 Oli_2322 Oli_1944 Oli_1945 Oli_2079 Oli_2090 Oli_1946 Oli_1947 Oli_2091 Oli_1948 Oli_1949 Oli_2092 Oli_1952 Oli_1953 Oli_2093 5'-CATATTTTCCGCTAAAACCGGTACCCTGACAAG-3' 5'-CTTGTCAGGGTACCGGTTTTAGCGGAAAATAT-3' 5'-TTAGGATCCTTATGTCTTCTTCTAACACGCC-3' 5'-TTAGAGCTCCTCGAGTTAACGACCTCCAGGAATTCTG-3' 5'-TTAACTCGAGGGACGACCTCCAGGAATTCTG-3' 5'-CGCGGATCCATGGCAAGAGGTAGTCATCACCAT CACCAT CACTCTTCTTCTAACACGCCATC-3' 5'-TTAAGATCTTTATGATGGAAGTGGAAGGATCG-3' 5'-TTAGAGCTCCTCGAGTCAACTTGAAAGGCTTTTCTTG-3' 5'-GGAAGATCTATGGCAAGAGGTAGTCATC ACCATCACC ATCACGAAGTGGAAGGATCGATGAAT-3' 5'-TTAGGATCCTTATGAGTTCCAATACGGCGTCG-3' 5'-TTAGAGCTCGTCGACTTACCGACCTCCAGGATTTC-3' 5'-CGCGGATCCATGGCAAGAGGTAGTCATCACC ATCACCA TCACAGTTCCAATACGGCGTCGTCC-3' 5'-TTAGGATCCTTATGAGTTCCACCGCGGCATCTTC-3' 5'-TTAGAGCTCCTCGAGTTACTGTAAACCTCCAGCACTTC-3' 5'-CGCGGATCCATGGCAAGAGGTAGTCATCAC CATCACCA TCACAGTTCCACCGCGGCATCTTCC-3'

10 DRS2 DNF1 AHA phi ProALA3 ProALIS1 ALA3 (Stable transformation) Oli_2251 Oli_2252 Oli_2463 Oli_2464 Oli_2244 Oli_2097 Oli_2094 Oli_2095 Oli_2468 Oli_2469 Oli_2470 Oli_ a 1032brs 5'-CACCATGAATGACGACAGAAACC-3' 5'-TCATATATCAAATGAAATATCATCTCTCG-3' 5'-CACCATGTCTGGAACTTTTCATGGCGATGGGC-3' 5'-TTAATTGTTCTGTTGTGTTCCGATTAAGGAGGC-3' 5'-TTAAGGATCCAGATGTCGAGTCTCGAAG-3' 5'-TTAACCGCGG CTACACAGTGTAGTGACTGGGAGT-3' 5'-GGATCCCAATGGCGGCACCACCAGCATCATCCTC-3' 5'-TTCCGCGGTAAAGTCGACGGGATCTCCTTCTGTTC-3' 5'-CACCATGGATTCTGAAATTCCTGGCTGTGGTC-3' 5'-GGATCCTTAGGAGGAATTCACACCGATCCA-3' 5'-CACCATGGATCATCTCTGATCTCCTCTCTC-3' 5'-GGATCCAGCTAATAAGAAAGGGAGATCTCTG-3' 5'-AAAGGCGCGCCACTCGAGATGGTTCGATCGGGTAGTTT-3' 5'-TTTGCGGCCGCTTACTTCTTCGGTACCTTTGGCC-3'

11 Supplemental Table 4. Plasmids constructed in this work Name Backbone Inserted genes FUNCTIONAL COMPLEMENTATION STUDIES pmp2033 pentr /D-TOPO pmp2126 pentr /D-TOPO pmp2393 pentr /D-TOPO pmp2371 pentr /D-TOPO pmp2642 pentr /D-TOPO pmp1965 prs423-gal pmp1982 prs423-gal pmp1983 prs423-gal pmp1984 prs423-gal pmp2027 prs423-gal pmp1999 prs423-gal pmp2000 prs423-gal pmp2001 prs423-gal pmp2022 prs423-gal pmp2038 prs423-gal pmp2039 prs423-gal pmp2040 prs423-gal pmp2041 prs423-gal pmp2072 prs423-gal ALA3 HA-ALA3 HA-ala3D413A DRS2 DNF1 Gateway Reading Frame Cassette A ALIS1+ Gateway Reading Frame Cassette A ALIS2+ Gateway Reading Frame Cassette A ALIS3+ Gateway Reading Frame Cassette A ALIS5+ Gateway Reading Frame Cassette A ALIS1 ALIS2 ALIS3 ALIS5 ALA3 ALA3+ALIS1 ALA3+ALIS2 ALA3+ALIS3 ALA3+ALIS5

12 pmp2127 prs423-gal pmp2128 prs423-gal pmp2129 prs423-gal pmp2130 prs423-gal pmp2151 prs423-gal pmp2531 prs423-gal pmp2152 prs423-gal pmp2532 prs423-gal pmp2153 prs423-gal pmp2154 prs423-gal pmp2155 prs423-gal pmp2367 prs423-gal pmp2646 prs423-gal RGSH 6 -ALIS1 RGSH 6 -ALIS2 RGSH 6 -ALIS3 RGSH 6 -ALIS5 HA-ALA3 HA-ala3D413A HA-ALA3+RGSH 6 -ALIS1 HA-ala3D413A+RGSH 6 -ALIS1 HA-ALA3+RGSH 6 -ALIS2 HA-ALA3+RGSH 6 -ALIS3 HA-ALA3+RGSH 6 -ALIS5 DRS2 DNF1 PROTEIN-PROTEIN INTERACTION STUDIES pmp2162 prs314- CUP -Nub pmp2368 pentr 11 pmp2142 prs314- CUP -Nub pmp2143 prs314- CUP -Nub pmp2135 pste14-cub-rura pmp2139 pste14-cub-rura Gateway Reading Frame Cassette A AHA2 AHA2 ALA3 ALIS phi RESCUE AND SUBCELLULAR LOCALIZATION STUDIES in planta pmp2197 pentr 11 ALIS1 w/o STOP

13 pmp2202 pentr 11 pmp2350 pentr /D-TOPO pmp2614 pmdc84 pmp2615 pmdc43 pmp2612 pmdc84 pmp2617 pmdc43 ALIS1 ALA3 w/o STOP ALA3:GFP GFP:ALA3 ALIS1:GFP GFP:ALIS1 ps1019 pgreenii TAP and 6xHis tags:gfp:ala3 TISSUE-SPECIFIC PROMOTER EXPRESSION STUDIES pmp2631 pentr 11 pmp2347 pmdc162 pmp2643 pentr 11 pmp2747 pmdc162 ProALA3 ProALA3:GUS ProALIS1 ProALIS1:GUS

Supplemental Figure 1. Mutation in NLA Causes Increased Pi Uptake Activity and

Supplemental Figure 1. Mutation in NLA Causes Increased Pi Uptake Activity and PHT1 Protein Amounts. (A) Shoot morphology of 19-day-old nla mutants under Pi-sufficient conditions. (B) [ 33 P]Pi uptake