Svp26 Facilitates Endoplasmic Reticulum to Golgi Transport of a Set of Mannosyltransferases in Saccharomyces cerevisiae

Yoichi Noda,Koji Yoda

doi:10.1074/jbc.m109.086272

Abstract

Svp26 is a polytopic integral membrane protein found in the ER and early Golgi compartment. In the Deltasvp26 cell, the Golgi mannosyltransferase Ktr3 remains in the ER. Here, we report that two other Golgi mannosyltransferases, Mnn2 and Mnn5 are also mislocalized and found in the ER in the absence of Svp26 and that localization of other mannosyltransferases including Mnn1 are not affected. Mnn2 and Mnn5 bind to Svp26 in vivo as Ktr3 does. Using an in vitro budding assay, the incorporation of Ktr3 and Mnn2 in the COPII vesicles is greatly stimulated by the presence of Svp26. As Svp26 itself is an efficient cargo, Svp26 is likely to support selective incorporation of a set of mannosyltransferases into COPII vesicles by working as their adaptor protein. The domain switching between Svp26-dependent Mnn2 or Ktr3 and Svp26-independent Mnn1 suggests that the lumenal domain of mannosyltransferases, but not the cytoplasmic or transmembrane domain, is responsible for recognition by Svp26.

Highlights

Recruitment of coatomer proteins, and the selected cargos are transported to the earlier compartments or to the ER [6, 7]
The first carbohydrates of glycoproteins are transferred from the dolichol intermediates to certain Asn or Ser/Thr residues of the precursor polypeptides in the ER, and further glycosyl residues are added or modified after moving from the ER to the Golgi [8]
The selective incorporation of the cargo proteins in the COPII and COPI vesicles should be very important for maintaining the specific protein composition of Golgi compartments

Summary

Genotype and plasmid

MATa ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa ⌬his3 ⌬leu2 ⌬trp1 ⌬ura3 ⌬svp26::kanMX4 MATa OCH1-3HA::HIS3 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa OCH1-3HA::HIS3 ⌬svp26::kanMX4 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa MNN1-3HA::LEU2 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa MNN1-3HA::LEU2 ⌬svp26::kanMX4 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa MNN5-3HA::HIS3 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa MNN2-3HA::LEU2 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa MNN5-3HA::HIS3 ⌬svp26::kanMX4 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa MNN5-3HA::HIS3 ⌬svp26::kanMX4 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa MNN2-3HA::LEU2 ⌬svp26::kanMX4 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura. As YNY729, pHI130 (SVP26-FLAG TRP1 CEN) MATa KTR3-3HA::LEU2 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura MATa KTR3-3HA::LEU2 ⌬svp26::kanMX4 ⌬his3 ⌬leu2 ⌬trp1 ⌬ura. As YNY762, pHI130 (SVP26-FLAG TRP1 CEN) MATa 3HA-SVP26::URA3 ⌬svp26::kanMX4 leu2-3, -112 ura MATa MNN2-3HA::LEU2 (multiple copies at LEU2 locus) MNN2-3HA::LEU2 (single copy at MNN2 locus) ⌬his3 ⌬trp1 ⌬ura. As YNY707, pHI130 (SVP26-FLAG TRP1 CEN) MATa KTR3 (NϩTM)-KTR3 (lumen)-3HA::URA3 ⌬svp26::LEU2 ⌬his3 ⌬leu2 ⌬trp ura3-52::kanMX4 MATa MNN1 (NϩTM)-MNN1 (lumen)-3HA::URA3 ⌬svp26::LEU2 ⌬his3 ⌬leu2 ⌬trp ura3-52::kanMX4 MATa KTR3 (NϩTM)-MNN1 (lumen)-3HA::URA3 ⌬svp26::LEU2 ⌬his3 ⌬leu2 ⌬trp ura3-52::kanMX4 MATa MNN1 (NϩTM)-KTR3 (lumen)-3HA::URA3 ⌬svp26::LEU2 ⌬his3 ⌬leu2 ⌬trp ura3-52::kanMX4 MATa MNN2 (NϩTM)-MNN1 (lumen)-3HA::URA3 ⌬svp26::LEU2 ⌬his3 ⌬leu2 ⌬trp ura3-52::kanMX4 MATa MNN2 (NϩTM)-MNN2 (lumen)-3HA::URA3 ⌬svp26::LEU2 ⌬his3 ⌬leu2 ⌬trp ura3-52::kanMX4 MATa MNN1 (NϩTM)-MNN2 (lumen)-3HA::URA3 ⌬svp26::LEU2 ⌬his3 ⌬leu2 ⌬trp ura3-52::kanMX4

Laboratory stock Laboratory stock

RESULTS

In the COPII vesicle formation system without adding the