Sec16 recruits Sec12 to transitional ER sites in Pichia pastoris.

摘要

In many organisms, COPII vesicles bud from a specialized domain of the ER known as the transitional ER (tER). Formation of the COPII vesicle coat starts with the exchange of GDP for GTP on the small GTPase Sar1, in a process catalyzed by the transmembrane protein Sec12. The localization of Sec12 varies between organisms. In the budding yeast Pichia pastoris, Sec12 is found at tER sites, while in the yeast Saccharomyces cerevisiae and in mammalian cells, Sec12 is found in the general ER. Previous studies indicated that both the lumenal and cytosolic domains of P. pastoris Sec12 are necessary for its localization to tER sites. Additionally, a partner protein is necessary to recruit Sec12 to tER sites.;This dissertation focuses on determining whether the peripheral membrane protein Sec16 is the partner protein that recruits P. pastoris Sec12 to tER sites. Sec16 was a good candidate because it functions in ER export and tER site organization, and because studies from another group revealed that Sec16 interacts with both Sec12 and the Sec12-like protein Sed4 in S. cerevisiae (Gimeno et al., 1995; P. Espenshade, personal communication). In this dissertation, I provide evidence that a C-terminal region of Sec16 interacts with the cytosolic domain of Sec12 in vitro, and that interfering with the Sec16/Sec12 interaction in vivo alters Sec12 localization. I showed that when Sec12 is overexpressed in P. pastoris, it is found in the general ER, but when Sec16 is also overexpressed, Sec12 remains at tER sites. However, if this experiment is performed with a C-terminally truncated version of Sec16, the overexpressed Sec12 is found in the general ER, indicating that a C-terminal region of Sec16 is needed to recruit Sec12 to tER sites. Additionally, when P. pastoris Sec12 (PpSec12) is expressed in S. cerevisiae, it is found in the general ER, but when P. pastoris Sec16 (PpSec16) is co-expressed with PpSec12 in S. cerevisiae, PpSec12 is found at tER sites. If a C-terminally truncated version of PpSec16 is expressed in S. cerevisiae together with PpSec12, PpSec12 is found in the general ER, mimicking the results seen with overexpression in P. pastoris.;Although viability is compromised by C-terminal truncations of Sec16, my data suggest that a strong Sec16/Sec12 interaction is not essential for life in budding yeasts. Nevertheless, I believe that this interaction is likely to be conserved. The tER localization of P. pastoris Sec12 may be an unusually clear consequence of the Sec16/Sec12 interaction. I speculate that Sec12 may regulate Sec16 function, or Sec16 may regulate Sec12 function, or both.