Insights into structural and regulatory roles of Sec16 in COPII vesicle formation at ER exit sites

Abstract

COPII-coated buds are formed at endoplasmic reticulum exit sites (ERES) to mediate ER-to-Golgi transport. Sec16 is an essential factor in ERES formation, as well as in COPII-mediated traffic in vivo. Sec16 interacts with multiple COPII proteins, although the functional significance of these interactions remains unknown. Here we present evidence that full-length Sec16 plays an important role in regulating Sar1 GTPase activity at the late steps of COPII vesicle formation. We show that Sec16 interacts with Sec23 and Sar1 through its C-terminal conserved region and hinders the ability of Sec31 to stimulate Sec23 GAP activity toward Sar1. We also find that purified Sec16 alone can self-assemble into homo-oligomeric complexes on a planar lipid membrane. These features ensure prolonged COPII coat association within a preformed Sec16 cluster, which may lead to the formation of ERES. Our results indicate a mechanistic relationship between COPII coat assembly and ERES formation.

FIGURE 1:

Sec16 localizes with COPII proteins at the ERES. (A) Fluorescent protein–tagged Sec16 is functional. sec16∆ cells expressing Sec16 from pRS316 (URA3) were transformed with pRS314 or pRS314-borne Sec16, or with C-terminally AcGFP- or mCherry-fused Sec16 (Sec16-AcGFP or Sec16-mCherry, respectively). Transformants were streaked on plates containing 5-FOA and incubated at 30°C. (B) Localization of Sec16 with COPII proteins. sec16∆ cells expressing Sec16-AcGFP with Sec13-mCherry, or Sec16-mCherry with Sar1-AcGFP, Sar1D32G-AcGFP, were grown to mid-log phase and observed by fluorescence microscopy. Arrowheads indicate Sar1-AcGFP–concentrated sites overlapping with Sec16-mCherry. (C) Sec16 L1089P mutant shows the temperature-sensitive phenotype in growth. sec16∆ cells expressing wild-type Sec16 or Sec16 P1089L mutant with or without AcGFP or mCherry fusion were streaked on plates and incubated at 23 and 37°C. (D) Sec16 L1089P mutant fails to localize at the ERES at 37°C. sec16∆ cells expressing Sec16-AcGFP or Sec16^L1089P-AcGFP were grown for 2 h at 23 or 37°C and observed by fluorescence microscopy. (E) The percentage of cells containing multiple ERES dots is indicated at 23 and 37°C. More than 100 cells were quantified in three individual experiments by fluorescence microscopy, and the error bars represent the SD. Scale bars, 4 μm.

FIGURE 2:

Inactivation of Sec16 alters localization of COPII proteins. sec16∆ cells expressing Sec13-mCherry with Sec16-AcGFP or Sec16^L1089P-AcGFP (A) or Sar1-AcGFP with Sec16-mCherry or Sec16^L1089P-mCherry (B) were grown for 2 h at the PT or the NPT and observed by fluorescence microscopy. Arrowheads indicate abnormal punctate structures of Sec16^L1089P-AcGFP and Sec13-mCherry. Scale bars, 4 μm.

FIGURE 3:

Sec16 negatively regulates the Sar1 GTPase activation of the COPII protein. (A) MBP-Sec16 and MBP-Sec16-mOrange are purified from yeast cells. Proteins from sec16∆ cells expressing MBP-Sec16 or MBP-Sec16-mOrange were purified by amylose resin and subjected to SDS–PAGE followed by Coomassie brilliant blue staining. (B) Activation of Sar1 GTPase was examined with Sec16. After preincubation of Sar1 (800 nM), GTP (100 μM), and synthetic liposomes (100 μg/ml) with or without MBP-Sec16 (70 nM) in the presence or absence of Sec13/31 (50 nM), Sec23/24 (50 nM) was added at 0 s to start the reaction. The subsequent decrease in tryptophan fluorescence signal was monitored over time at 340 nm. (C) Coat disassembly is monitored by light scattering assay. Light scattering signals represent the state of the coat-assembled/disassembled liposomes. After preincubation of Sar1 (800 nM), GTP (100 μM), and liposomes (100 μg/ml) with Sec13/31 (75 nM), Sec23/24 (75 nM) was injected into the mixture at 0 s. Where indicated, MBP-Sec16 (60 nM) was added in the starting mixture. The subsequent decrease in change of light scattering signal was monitored over time. The data represent the average of three independent experiments.

FIGURE 4:

Mapping of the binding regions of Sec23 and Sec31 within Sec16. (A) The effect of Sec16 truncations on cell growth. A schematic drawing of Sec16 is shown indicating the location of two conserved domains. The ability of each Sec16 truncation mutant to support the growth of sec16∆ cells was tested on 5-FOA plates as described in the legend of Figure 1A. (B) Mapping of the Sec31-binding region within Sec16 by a yeast two-hybrid assay. The PJ69-4A strain was transformed with plasmids containing the binding domain (BD)–fused Sec31 and the activation domain (AD)–fused Sec16 fragment, and transformants were grown on −histidine plates at 30ºC for 5 d. (C) Sec31 interacts with the 501–560 region of Sec16. MBP and indicated MBP-Sec16 purified from E. coli were immobilized on amylose resin and incubated with Sec13/31. Affinity-isolated proteins were subjected to SDS–PAGE and stained with Sypro Orange. (D) Sec16 lacking the Sec31-binding region does not fully support growth of sec16∆ cells. The ability of Sec16 lacking the 501–560 region to support the growth of sec16∆ cells was tested on 5-FOA plates as described in the legend of Figure 1A. (E) The Sec31-binding region is important for Sec16 function to facilitate ER exit of secretory proteins. Total cell extracts of sec16∆ cells expressing wild-type Sec16 or Sec16^∆501-560 were separated by SDS–PAGE followed by immunoblotting with anti-CPY or anti-Pgk1 antiserum. (F) Yeast two-hybrid assay between Sec23 and the C-terminal region of Sec16. The Pj69-4A strain was transformed with plasmids containing BD-fused Sec23 and AD-fused Sec16 fragments, and transformants were grown on −histidine plates at 30ºC for 5 d. (G) Sec23 interacts with the C-terminal region including the CTCD of Sec16. MBP and indicated MBP-Sec16 purified from E. coli were immobilized on amylose resin and incubated with Sec23/24. Affinity-isolated proteins were subjected to SDS–PAGE and stained with Sypro Orange. Sec24 and MBP-Sec16^1639-1967 have roughly the same apparent mobility on the gel.

FIGURE 5:

The CTCD is critical for Sec16 function. (A) The ability of PpSec16 to support growth of sec16∆ cells was tested on 5-FOA plates as described in the legend of Figure 1A. (B) Total cell extracts of S. cerevisiae cells (SEY6210) expressing HA-tagged S. cerevisiae (Sc), P. pastoris (Pp), or chimeric Sec16 indicated were separated by SDS–PAGE followed by immunoblotting with anti-HA or anti-Pgk1 antiserum. Chimeras examined are depicted in D. (C) PpSec16 localizes at the ERES in S. cerevisiae cells. S. cerevisiae cells (SEY6210) expressing PpSec16-AcGFP with Sec13-mCherry were grown to mid-log phase and examined by fluorescence microscopy. Scale bar, 4 μm. (D) Chimeras between S. cerevisiae (ScSec16) and P. pastoris (PpSec16) Sec16. A schematic diagram shows chimeric forms of Sec16. The sequence derived from PpSec16 is depicted as black and light gray bars, and the sequence from ScSec16 is shown as white and dark gray bars. Gray bars display the CDC and CTCD. PS-1Sec16; PpSec16^1-1010 and ScSec16^960-2195, PS-2Sec16; PpSec16^1-1461 and ScSec16^1423-2195, PS-3Sec16; PpSec16^1-2266 and ScSec16^1895-2195, PS-4Sec16; PpSec16^1-2361 and ScSec16^1991-2195, SPSec16; and ScSec16^1-1996 and PpSec16^2369-2550. The ability of each Sec16 chimera to support the growth of sec16∆ cells was tested on 5-FOA plates as described in the legend of Figure 1A.

FIGURE 6:

The CTCD alters the interaction between Sec23 and Sec31. (A) The GTPase activity of Sar1 was examined with MBP-Sec16^1639-2195 (150 nM) as in Figure 3B. Where indicated, Sec13/31 was added in the starting mixture. (B) Sar1 GTPase activity was monitored with the indicated concentrations of MBP-Sec16^1639-2195 in the presence of Sec13/31 as in A. (C) Sar1 GTPase activity was monitored with indicated MBP-Sec16 in the presence of Sec13/31 as in A. (D) Liposome-binding assay of COPII proteins with MBP-Sec16^1639-2195. Sar1 (800 nM), Sec23/24 (100 nM), Sec13/31 (150 nM), and MBP-Sec16^1639-2195 (300 nM) were incubated with GDP or GMP-PNP (100 μM) and synthetic liposomes (100 μg/ml) subjected to flotation on a sucrose density gradient. Float fractions were subjected to SDS–PAGE and stained with Sypro Orange.

FIGURE 7:

Sec16 self-assembles on the membrane. (A) Sec16 self-assembles. Salt extracts of sec16∆ cells simultaneously expressing MBP-Sec16 and Sec16-AcGFP were incubated with amylose resin. Total lysates and eluted proteins were subjected to SDS–PAGE followed by immunoblotting with anti-MBP or anti-GFP antibody. (B) Imaging of MBP-Sec16-mOrange molecules on a planar lipid bilayer. Right, the system for observing the horizontal planar lipid membrane under a microscope. An artificial planar lipid bilayer was formed across the hole on the bottom of the upper chamber and observed under evanescent field illumination before and after injection of MBP-Sec16-mOrange (200 pM). Scale bar, 10 μm. (C) Distribution of the fluorescence intensity of MBP-Sec16-mOrange on the membrane. A histogram of the fluorescence intensity was fitted to a single-Gaussian distribution (gray curve). (D) Intensity profile showing multiple-step photobleaching. Two representative examples are shown in which four bleaching steps are clearly discernible.