Distinct adaptor proteins assist exit of Kre2-family proteins from the yeast ER

Abstract

The Svp26 protein of S. cerevisiae is an ER- and Golgi-localized integral membrane protein with 4 potential membrane-spanning domains. It functions as an adaptor protein that facilitates the ER exit of Ktr3, a mannosyltransferase required for biosynthesis of O-linked oligosaccharides, and the ER exit of Mnn2 and Mnn5, mannosyltransferases, which participate in the biosynthesis of N-linked oligosaccharides. Ktr3 belongs to the Kre2 family, which consists of 9 members of type-II membrane proteins sharing sequence similarities. In this report, we examined all Kre2 family members and found that the Golgi localizations of two others, Kre2 and Ktr1, were dependent on Svp26 by immunofluorescence microscopy and cell fractionations in sucrose density gradients. We show that Svp26 functions in facilitating the ER exit of Kre2 and Ktr1 by an in vitro COPII budding assay. Golgi localization of Ktr4 was not dependent on Svp26. Screening null mutants of the genes encoding abundant COPII membrane proteins for those showing mislocalization of Ktr4 in the ER revealed that Erv41 and Erv46 are required for the correct Golgi localization of Ktr4. We provide biochemical evidence that the Erv41-Erv46 complex functions as an adaptor protein for ER exit of Ktr4. This is the first demonstration of the molecular function of this evolutionally conserved protein complex. The domain switching experiments show that the lumenal domain of Ktr4 is responsible for recognition by the Erv41-Erv46 complex. Thus, ER exit of Kre2-family proteins is dependent on distinct adaptor proteins and our results provide new insights into the traffic of Kre2-family mannosyltransferases.

Both Svp26 and Mnn6 are required for the efficient ER exit of Mnn4 in <i>Saccharomyces cerevisiae</i>

“…Localization of Mnn6-myc was also observed by indirect immunofluorescent microscopy. Mnn6-myc showed an image of ER-localized proteins, both in the wild-type and Δsvp26 cells, which is consistent with our previous result shown by sucrose density fractionation (Noda et al, 2014).…”

“…Deletion of either the SVP26 gene or the MNN6 gene leads to the accumulation of Mnn4 in the ER Mnn6 belongs to the Kre2 family proteins, and, unlike other members of the Kre2 family which localize to the Golgi, probably localizes to the ER as previously suggested 5 by our sucrose density fractionation experiment (Noda et al, 2014). It has been shown that in addition to Mnn6, Mnn4, which is probably also a type II membrane protein, is required for mannosyl phosphate transfer to the glycans (Odani et al, 1996).…”

“…ER adaptor proteins in S. cerevisiae, including Svp26, facilitate ER exit of many proteins destined for the intracellular organelles or periplasmic space (Dancourt and Barlowe, 2010;Noda and Yoda, 2013). ER exit of the 7 proteins, Ktr3, Ktr1, Kre2, Mnn2, Mnn5, Pho8 and Gda1, has been previously reported to be dependent on Svp26 (Anand et al, 2009;Bue et al, 2006;Inadome et al, 5 2005;Noda and Yoda, 2010;Noda et al, 2014). In addition, we recently identified Mnt2 and Mnt3 proteins as new cargos of Svp26 (in press).…”

“…Because Svp26 functions as an ER exit adaptor protein for Golgi mannosyltransferases involved in either O-glycosylation or N-glycosylation (Anand et al, 2009;Bue et al, 2006;Inadome et al, 2005;Noda and Yoda, 2010;Noda et al, 2014), we considered the possibility that Svp26 is involved also in the ER exit of Mnn4. We first examined if 15 deletion of the SVP26 gene or the MNN6 gene affects the localization of Mnn4.…”

“…Golgi localized type II membrane proteins and their ER exit is dependent on Svp26, which functions to facilitate an ER exit of a set of Golgi-localized mannosyltransferases (Inadome et al, 2005;Noda et al, 2014;Noda and Yoda, 2010), including Mnn2 and Mnn5. It was also reported that an ER exit of Och1 and Mnn1, other mannosyltransferases involved in the N-glycan modification, was not dependent on 30…”