1. The Transitional ER Localization Mechanism of Pichia pastoris Sec12
Jon Soderholm, Dibyendu Bhattacharyya, Daniel Strongin, Vida Markovitz, Pamela L Connerly, Catherine A Reinke, Benjamin S Glick 
Developmental Cell 
Volume 6, Issue 5, Pages (May 2004)
DOI: /S (04)

2. Figure 1
P. pastoris Sec12 Rapidly Exchanges between tER Sites and the General ER
A strain was constructed in which the endogenous P. pastoris SEC12 gene was deleted and GFP-SEC12 was expressed from the SEC12 promoter (see Experimental Procedures). We found that a Sec12 fusion to one or three copies of the standard enhanced GFP (EGFP; Cormack et al., 1996) yielded virtually no fluorescence, but a fusion to three tandem copies of an alternative GFP variant (sGFP; Kahana et al., 1998) yielded a good signal.
(A) 4D confocal microscopy was used to examine the dynamics of GFP-Sec12 in living P. pastoris cells. Shown are selected panels from Supplemental Movie S1 ( with times indicated in min:s format. The arrowhead points to a GFP-Sec12-labeled tER site that was bleached and then allowed to recover fluorescence. Scale bar, 2 μm.
(B) Fluorescence recovery after photobleaching was quantified for the tER site marked in (A). The horizontal axis indicates the time in seconds after the photobleaching, and the vertical axis indicates fluorescence intensity in arbitrary units. The half-time of fluorescence recovery for this tER site was approximately 60 s. Bleaching of this tER site resulted in a loss of approximately 65% of the total cellular fluorescence, and as a result, the tER site recovered only about 35% of its original fluorescence.
Developmental Cell 2004 6, DOI: ( /S (04) )

3. Figure 2
The Association of P. pastoris Sec12 with tER Sites Is Saturable
P. pastoris cells were grown to log phase in SYG, washed, then transferred to SYM for various times to induce expression from the AOX1 promoter. The cells were processed for immunofluorescence with anti-GluGlu antibody either immediately following the switch to SYM (t = 0 hr), after 4 hr of growth in SYM (t = 4 hr), or after 8 hr of growth in SYM (t = 8 hr). Sec12-GG, GluGlu-tagged Sec12 in which the C-terminal sequence EGENINHDEL was replaced by EGENINEEEEYMPMEEALHDEL. OEx, overexpression. Scale bar, 2 μm.
(A–C) The chromosomal copy of SEC12 was replaced with SEC12-GG under control of the endogenous SEC12 promoter. These cells were transformed with the parental AOX1 promoter vector pIB4.
(D–F) The chromosomal copy of SEC12 was replaced with SEC12-GG under control of the endogenous SEC12 promoter. These cells were transformed with a derivative of pIB4 encoding untagged P. pastoris Sec12 under control of the AOX1 promoter.
(G–I) P. pastoris cells containing a wild-type chromosomal copy of SEC12 were transformed with a vector encoding Sec12-GG under control of the AOX1 promoter. All of the cells were photographed with an identical exposure time, except that the exposure time for (I) was shorter because the fluorescence signal was very strong.
Developmental Cell 2004 6, DOI: ( /S (04) )

4. Figure 3
P. pastoris Sec12 Contains a tER Localization Signal that Functions in P. pastoris but Not in S. cerevisiae
(A) Either P. pastoris Sec12-GG or S. cerevisiae Sec12-GG was expressed from the S. cerevisiae SEC12 promoter in an S. cerevisiae strain lacking the endogenous chromosomal copy of SEC12. The tagged proteins were visualized by immunofluorescence. In each case, cells are shown expressing low (left panels), moderate (middle panels), or high (right panels) levels of Sec12-GG. The contrast of these images was adjusted to facilitate comparison.
(B) Either S. cerevisiae Sec12-GG or P. pastoris Sec12-GG was expressed from the AOX1 promoter in a P. pastoris strain that also contained untagged endogenous Sec12. Cells were processed for immunofluorescence after 4 hr of growth in SYM. As shown in Figure 2, after 4 hr of AOX1-driven expression, Sec12 protein levels are too low to saturate tER localization. Nevertheless, in this and subsequent experiments employing the AOX1 promoter, we carefully examined cells exhibiting varying levels of expression to ensure that a general ER pattern was not due to overexpression. Scale bar, 2 μm.
Developmental Cell 2004 6, DOI: ( /S (04) )

5. Figure 4
tER Localization of P. pastoris Sec12 Requires Both the Cytosolic and Lumenal Domains
Chimeric or truncated forms of P. pastoris Sec12 were expressed in P. pastoris cells and examined by immunofluorescence. Each construct included a GluGlu tag, either at the C terminus or just upstream of the HDEL tetrapeptide (see Figure 2 legend).
(A) Schematic diagram of Sec12 from P. pastoris (“P.p.,” orange) and S. cerevisiae (“S.c.,” blue). The lumenal domain of P. pastoris Sec12 includes a basic segment (“B;” residues 360–598), a central segment (“C;” residues 599–950), an acidic segment (“A;” residues 951–1034), and a C-terminal HDEL tetrapeptide.
(B) Left panel: the cytosolic domain of P. pastoris Sec12 (residues 2–328) was removed. This construct was expressed from the SEC12 promoter in a P. pastoris strain containing a truncated Sec12 that lacked the lumenal domain. Right panel: the lumenal domain of P. pastoris Sec12 (residues 367–1038) was removed. This construct was made by gene replacement at the SEC12 locus, and to boost expression to detectable levels, a second copy of the same truncated gene was expressed from the AOX1 promoter with 4 hr of induction in SYM. Note that the nuclei tend to be smaller in methanol-grown P. pastoris cells than in glucose-grown cells. Scale bar, 2 μm.
(C) Left panel: the cytosolic domain of P. pastoris Sec12 (residues 1–332) was replaced with the cytosolic domain of S. cerevisiae Sec12 (residues 1–349). This construct was expressed from the AOX1 promoter with 4 hr of induction in SYM, in a P. pastoris strain containing a truncated Sec12 that lacked the lumenal domain. Middle panel: the transmembrane domain of P. pastoris Sec12 (residues 338–359) was replaced with the transmembrane domain of S. cerevisiae Sec12 (residues 355–373). This construct was expressed from the SEC12 promoter in a sec12Δ strain of P. pastoris. Right panel: the lumenal domain of P. pastoris Sec12 (residues 360–1038) was replaced with the lumenal domain of S. cerevisiae Sec12 (residues 374–471). This construct was made by gene replacement at the SEC12 locus, and to boost expression to detectable levels, a second copy of the same chimeric gene was expressed from the AOX1 promoter with 4 hr of induction in SYM.
Developmental Cell 2004 6, DOI: ( /S (04) )

6. Figure 5
The Lumenal Domain of P. pastoris Sec12 Contains Redundant tER Localization Signals
(A) The endogenous SEC12 locus in P. pastoris was replaced with a series of constructs encoding the indicated deletions of segments of the Sec12 lumenal domain (see Figure 4A for segment descriptions). Each construct included a GluGlu tag, either at the C terminus or just upstream of the HDEL tetrapeptide (see Figure 2 legend). For the ΔB,A,HDEL construct and the ΔB,C construct, expression was boosted to detectable levels by expressing a second copy of the same mutant gene from the AOX1 promoter with 4 hr of induction in SYM. The localization of the mutant proteins was analyzed by immunofluorescence.
(B) Representative immunofluorescence images are shown for the ΔC,A,HDEL construct (left panel), the ΔB,A,HDEL construct (middle panel), and the ΔB,C construct (right panel). The punctate structures seen with the ΔC,A,HDEL and ΔB,C constructs also labeled with Sec13-GFP, confirming that these structures are tER sites (not shown). Scale bar, 2 μm.
Developmental Cell 2004 6, DOI: ( /S (04) )

7. Figure 6
Oligomerization of the Lumenal Domain Promotes tER Localization of P. pastoris Sec12
(A) Shown at the top is the sequence of the Gcn4 leucine zipper. The schematic diagrams represent mutant versions of P. pastoris Sec12 in which the leucine zipper (small white rectangle) replaced either the lumenal domain in the construct Sec12-LZ or the cytosolic domain in the construct LZ-Sec12. In Sec12-LZ(L19P), the leucine in the Gcn4 sequence at position 19 (arrow) was changed to a proline. Each of these proteins had a C-terminal GluGlu tag.
(B) Immunofluorescence of cells containing Sec12-LZ (left panel) or Sec12-LZ(L19P) (right panel). Each construct was made by gene replacement, and to boost expression to detectable levels, a second copy of the same mutant gene was expressed from the AOX1 promoter with 4 hr of induction in SYM.
(C) Immunofluorescence of cells containing LZ-Sec12. This construct was expressed from the SEC12 promoter in a P. pastoris strain containing a truncated Sec12 that lacked the lumenal domain. Scale bar, 2 μm.
(D) Yeast two-hybrid analysis using a URA3 “bait” vector and a LEU2 “prey” vector (James et al., 1996). Lane 1: empty bait and prey vectors. Lane 2: Bait and prey vectors both expressing fusions to the basic segment of the P. pastoris Sec12 lumenal domain. Lane 3: Empty prey vector with a bait vector expressing a Sec12 basic segment fusion. Lane 4: Empty bait vector with a prey vector expressing a Sec12 basic segment fusion. Cells growing on minimal medium lacking leucine (leu) and uracil (ura) were patched either on the same medium (left panel) or on media that also lacked histidine (his; middle panel) or adenine (ade; right panel), and were incubated at 30°C for 3 days.
Developmental Cell 2004 6, DOI: ( /S (04) )

8. Figure 7
Delocalization of Sec12 Does Not Perturb the Localization of Downstream tER or Golgi Components
tER and Golgi organization was examined by immunofluorescence and electron microscopy in P. pastoris strains containing either tER-localized wild-type Sec12, or a delocalized chimeric Sec12 in which the lumenal domain had been replaced with that of S. cerevisiae Sec12 (see Figure 4C). This chimeric Sec12 was expressed from the endogenous SEC12 promoter. The strains used in (A) and (B) also expressed Sec13-GFP.
(A) Double label immunofluorescence of the tER markers Sar1 and Sec13-GFP in cells containing either wild-type Sec12 (top panels) or delocalized chimeric Sec12 (bottom panels). Scale bar, 2 μm.
(B) Double label immunofluorescence of the tER marker Sec13-GFP and the Golgi marker Och1-HA in cells containing either wild-type Sec12 (top panels) or delocalized chimeric Sec12 (bottom panels).
(C) Representative electron micrographs of P. pastoris cells containing either wild-type Sec12 (left panel) or delocalized chimeric Sec12 (right panel). Electron microscopy was performed as previously described (Rossanese et al., 1999). N, nucleus. Scale bars, 0.25 μm.
Developmental Cell 2004 6, DOI: ( /S (04) )