Sec12 Binds to Sec16 at Transitional ER Sites

COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12–Sec16 interaction has a conserved role in ER export

Sec12 Binds to Sec16 at Transitional ER Sites

COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12–Sec16 interaction has a conserved role in ER export.</p

Sec16 is a Determinant of Transitional ER Organization

SummaryBackground: Proteins are exported from the ER at transitional ER (tER) sites, which produce COPII vesicles. However, little is known about how COPII components are concentrated at tER sites. The budding yeast Pichia pastoris contains discrete tER sites and is, therefore, an ideal system for studying tER organization.Results: We show that the integrity of tER sites in P. pastoris requires the peripheral membrane protein Sec16. P. pastoris Sec16 is an order of magnitude less abundant than a COPII-coat protein at tER sites and seems to show a saturable association with these sites. A temperature-sensitive mutation in Sec16 causes tER fragmentation at elevated temperature. This effect is specific because when COPII assembly is inhibited with a dominant-negative form of the Sar1 GTPase, tER sites remain intact. The tER fragmentation in the sec16 mutant is accompanied by disruption of Golgi stacks.Conclusions: Our data suggest that Sec16 helps to organize patches of COPII-coat proteins into clusters that represent tER sites. The Golgi disruption that occurs in the sec16 mutant provides evidence that Golgi structure in budding yeasts depends on tER organization

Viability of <i>S. cerevisiae</i> cells carrying <i>PpSEC12</i> as the only gene from the <i>SEC12</i> family.

<p>A plasmid shuffle was performed in <i>sed4</i>Δ <i>sec12</i>Δ cells, with <i>SED4</i> in a <i>URA3</i> plasmid plus either <i>ScSEC12</i> (top row) or <i>PpSEC12</i> (middle row) in a <i>LEU2</i> plasmid. Both strains grew on rich media (YPD) and also on media containing 5-FOA, indicating that <i>PpSEC12</i> could replace <i>ScSEC12</i> even in the absence of <i>SED4</i>. As a control, <i>sec12</i>Δ cells carrying <i>ScSEC12</i> on a <i>URA3</i> plasmid were plated on the same media, and no growth was seen in the presence of 5-FOA.</p

Requirement of the C-terminal portion of PpSec16 for tER localization of PpSec12 in both <i>P. pastoris</i> and <i>S. cerevisiae</i>.

<p>(A) As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g001" target="_blank">Fig. 1</a>, <i>P. pastoris</i> cells expressed PpSec12-GG from the endogenous promoter plus untagged Sec12 from the <i>AOX1</i> promoter, resulting in a high total level of PpSec12 expression. In the same cells, a truncated version of PpSec16 lacking residues 1967–2550 was tagged with GFP and overexpressed as a second copy using the <i>AOX1</i> promoter. PpSec16(Δ1967–2550)-GFP was found in punctate tER sites. By contrast to the result obtained when full-length PpSec16 was expressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g001" target="_blank">Fig. 1</a>), PpSec12-GG was found in the general ER. (B) <i>S. cerevisiae</i> cells expressed the same truncated version of PpSec16 as in (A), except that the protein was tagged with YFP and was expressed under control of the <i>GAL10</i> promoter. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g002" target="_blank">Fig. 2B</a>, ScSec13-CFP and PpSec12-GG were also expressed in these cells. By contrast to the result obtained when full-length PpSec16 was expressed (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g002" target="_blank">Fig. 2B</a>), PpSec12-GG was found in the general ER. Scale bars, 2 µm.</p

Colocalization of mammalian Sec12 with Sec16A at tER sites.

<p>(A) U2OS human osteosarcoma cells were subjected to immunofluorescence as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone.0031156-Bhattacharyya2" target="_blank">[28]</a> using commercial antibodies against human Sec12 and Sec16A. Scale bar, 2 µm. (B) Plasmids encoding YFP-tagged full-length human Sec12 and CFP-tagged full-length Sec16B were co-transfected into U2OS cells. The cells were imaged at 16 h post-transfection, a time point that yielded relatively low expression levels. Scale bar, 2 µm. (C) HeLa cells were transfected where indicated with plasmids encoding either monomeric GFP fused to a C-terminal region (“CTR”) of human Sec16A (residues 1909–2332), or an N-terminally triple-FLAG-tagged cytosolic domain (“Cyt”) of human Sec12 (residues 1–386). At 24 h post-transfection, the cells were lysed and the lysate was subjected to immunoprecipitation (“IP”) with anti-FLAG antibody. The immunoprecipitated material and 5% of the lysate (“5% Input”) was subjected to SDS-PAGE followed by immunoblotting with either anti-FLAG or anti-GFP antibody.</p

Effect of deleting nonessential PpSec16 regions on PpSec12 localization in <i>P. pastoris</i>.

<p>(A) Diagram of the domain organization of PpSec16. Shading indicates conserved regions while hatch marks indicate an essential region. CCD, central conserved domain; Q, glutamine-rich region; CTR, C-terminal conserved region. Deletions introduced by gene replacement are indicated. None of these deletions affected PpSec12 localization. (B) Representative images of PpSec12-GG localization in <i>P. pastoris</i> cells carrying the indicated deletions in PpSec16. Scale bar, 2 µm.</p

Biochemical interaction of the C-terminal portion of PpSec16 with the cytosolic domain of PpSec12.

<p>Glutathione-agarose beads were incubated with a bacterial lysate from cells expressing either GST alone, or GST fused to the C-terminal residues 1960–2550 of PpSec16. Sufficient lysate was used to saturate the binding sites on the glutathione-agarose. A second incubation was then performed with sub-saturating amounts of a bacterial lysate from cells expressing a hexahistidine-tagged version of the cytosolic domain of PpSec12 (PpSec12(cyto)-His6). The beads were centrifuged, and the unbound material in the supernatant was collected. Bound protein was eluted from the beads with 100 mM glutathione. I, input (100% relative to other lanes); U, unbound; B, bound. PpSec12(cyto)-His6 bound to the beads carrying GST-PpSec16(1960–2550) but not to the beads carrying GST alone.</p

Requirement of the C-terminal portion of PpSec16 for recruiting overexpressed PpSec12 to tER sites.

<p>As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031156#pone-0031156-g001" target="_blank">Fig. 1</a>, PpSec16-GFP was overexpressed in <i>P. pastoris</i> cells overexpressing PpSec12, except that deletions were introduced as indicated near the C-terminus of PpSec16. Two hundred randomly chosen cells from each of the indicated <i>P. pastoris</i> strains were examined by immunofluorescence and scored for colocalization of PpSec12-GG with PpSec16-GFP. Cells in which nearly all of the PpSec12-GG overlapped with PpSec16-GFP were scored as having strong colocalization (+). Cells in which PpSec12-GG showed clear concentration in the PpSec16-GFP puncta but also showed prominent staining outside of these puncta were scored as having partial colocalization (+/−). Cells showing no visible concentration of PpSec12-GG in the PpSec16-GFP puncta were scored as having no colocalization (−). Colocalization was virtually abolished by deleting the entire C-terminal portion of PpSec16, and was strongly reduced by deleting only the C-terminal conserved region (CTR).</p

Recruitment of overexpressed PpSec12 to tER sites in <i>P. pastoris</i> by simultaneous overexpression of PpSec16.

<p>PpSec12 was tagged with the Glu-Glu epitope (PpSec12-GG) by gene replacement, and a second untagged copy of PpSec12 was expressed in the same cells using the methanol-inducible <i>AOX1</i> promoter, resulting in a high total level of PpSec12 expression. Top row: in a strain overexpressing PpSec12, PpSec16 was expressed at normal levels after being tagged by gene replacement with GFP. A small fraction of the PpSec12-GG colocalized with PpSec16-GFP, but most of the PpSec12-GG was in the general ER as indicated by the prominent nuclear envelope signal. Bottom row: in a strain overexpressing PpSec12, PpSec16-GFP was overexpressed as a second copy using the <i>AOX1</i> promoter. Most of the PpSec12-GG colocalized with PpSec16-GFP in exaggerated tER sites. Scale bar, 2 µm.</p