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Noda Institute for Scientific Research GRANT2013 Research Grant

Investigation of the molecular mechanisms regulating thesecretion from the endoplasmic reticulum in SaccharomycescerevisiaeKen SatoDepartment of Life Sciences, The University of Tokyo

Research aimsIn eukaryotic cells, secretory pathways serve as themajor routes for extracellular protein transport, and manyquestions need to be yet answered. Although the secretorypathway is initiated in the endoplasmic reticulum (ER), anumber of its aspects, such as the molecular mechanisms ofcargo selection and regulation, remain unclear. The cytosolic coat protein complex II (COPII) is required for thephysical deformation of the ER membrane, and in combination with the small GTPase Sar1, it drives the formationof the COPII vesicles1) (Fig. 1). COPII vesicle formationoccurs at specialized membrane domains, the so-called ERexit sites (ERES), which are thought to be responsible forthe regulation of ER export.2) However, most of the molecular mechanisms underlying the assembly and regulation ofthe ERES remain unknown. In this study, we conducted acomprehensive investigation of the organization of theERES.
MethodsTo identify the functional domain of Sec16, which is responsible for its transport function, various deletion mutants of Sec16 were expressed and examined. To identifythe Sec16 domain required for ER localization and ERESorganization, the subcellular localization of these fragments

Fig. 1.

(fused to fluorescent proteins) was analyzed. In addition,subcellular localization and biochemical characterization ofthe Sec23 homolog, Nel1, was performed. Furthermore, toinvestigate the steady-state organization of ERES in livecells, fluorescence confocal microscopy was carried out oncells expressing Sec24-GFP paired with the expression ofone of the Sec24 isoforms tagged with mCherry, to determine whether the Sec24 isoforms are uniformly distributedat each ERES.
Results1)

Identification of the functional domain of Sec16We found that the central conserved domain (CCD) ofSec16, which is well conserved among the Sec16 homologsin various species, is responsible for its ER localization. Wealso show that the C-terminal domain of Sec16, in combination with the CCD domain, is responsible for the membrane anchoring of Sec16, via ER-localized Sar1-GTP. Inaddition, the N-terminal domain of Sec16 is required forthe functional assembly of the COPII coat in the ER.
2)

Functional analysis of Nel1We found that Nel1 overexpression cannot compensatefor the loss of Sec23 function and that Nel1 does not associate with Sec24 and its isoforms, indicating that Nel1 activity is not functionally redundant with Sec23. We show

COPII vesicle formation in the ER

46(Sec24 and Lst1) are unequally distributed at each ERES ata steady state, suggesting that each ERES has a distinctpreference for the cargo proteins. These results also suggestthat ERES in S. cerevisiae are heterogeneous with respectto their composition.
Conclusions

Fig. 2. NEL1 has a genetic interaction with SAR1. A) Growth phenotypeof the wild-type, nel1∆, and sar1D32G strains at 23°C. B) Growth phenotype of the sar1D32G strain and sar1D32G/NEL1∆ double mutant strainat 23°C.

that purified Nel1 exhibits strong Sar1 GAP activity, butdoes not bind to Sec13/31, suggesting that Nel1 plays a rolein the regulation of the GTPase activity of Sar1, but mayact distinctly from Sec23 as a subunit of the COPII coat.
We also demonstrate that the chromosomal deletion ofNEL1 leads to a significant growth defect in the temperature-sensitive sar1D32G background, suggesting a possiblefunctional link between these proteins.3)3) Analysis of the diversity of the ERESUsing live cell-imaging analyses in S. cerevisiae, wedemonstrate that the isoforms of cargo-adaptor subunits

Our results show that Sec16 is not only a scaffoldingprotein for COPII vesicle formation, but also serves to organize COPII coat assembly at ERES. We identified Nel1as a novel homolog of Sec23 that exhibits genetic and biochemical interactions with Sar1 in S. cerevisiae. ERES,which are visible as 50–100 small dots in S. cerevisiae, areheterogeneous with respect to their functional characteristics.
References1)

Sato, K. and Nakano, A. (2007) Mechanisms of COPII vesicle formation and protein sorting. FEBS Lett. 581: 2076–2082.
2) Yorimitsu, T., and Sato, K. (2012) Insights into structural and regulatory roles of Sec16 in COPII vesicle formation at ER exit sites. Mol.
Biol. Cell 23: 2930–2942.
3) Kodera, C., Yorimitsu, T., and Sato, K. (2014) Sec23 homolog Nel1is a novel GTPase-activating protein for Sar1 but does not functionas a subunit of the coat protein complex II (COPII) coat. J. Biol.
Chem. 289: 21423–21432.