43NISR Research GRANT
2009 Research Grant

Mechanisms of Golgi-localization of Mannosyltransferases
Yoichi NODA
Department of Biotechnology, University of Tokyo

Reseach aims
The Golgi apparatus comprises of multiple compartments with different components and functions. Thesecompartments are successively formed by the maturation of
the earliest compartment derived from the ER through homotypic fusion of the COPII vesicles (1). The maturation ofcompartments is believed to occur by removing the components of the earlier compartments and acquiring those of
the later ones in a COPI vesicle-dependent manner (2, 3).
Svp26 was discovered as a novel function-unknown protein in our global inspection of membrane proteins in theearly Golgi compartment (4). Svp26 is an integral membrane protein with 4 predicted transmembrane segments.
Although Svp26 is dispensable for viability of the yeast, wefound abnormal hyper-mannosylation of N-glycosyl chainsand mislocalization of Golgi mannosyltransferase Ktr3 to
the ER in the Dsvp26 disruptant cell. Immunoprecipitationfrom the cleared lysate using 1% Triton X-100 indicatedspecific binding between Ktr3 and Svp26. These results
suggest that Svp26 functions to ensure the correct localization of Ktr3 in the Golgi compartments. In this study, weaimed to reveal the molecular function of Svp26 and to discover new proteins that regulate the Golgi-localization of
mannosyltransferases the localization of which are not dependent on Svp26.
Methods
Construction of yeast strains expressing triple HA
tagged mannosyltransferase. For tagging mannosyltransferases with three copies of the HA epitope at their C-termini, an appropriate DNA fragment of the 3-region of eachmannosyltransferase was amplified by PCR and cloned in
pYN497 (HIS3 marker) or pYN503 (LEU2 marker). Theseplasmids carry a coding sequence for triple HA followed bya TDH3 terminator. The resulting plasmids were linearized
by cutting at a unique restriction site located within acloned region of each ORF, and used for the transformationof yeast to obtain desired strains with chromosomallytagged genes by homologous recombination.
In vitro COPII vesicle budding assay. Purification ofCOPII coat components Sar1, Sec23/24 and Sec13/31, andthe vesicle budding assay were performed as described previously. Microsomal membranes were prepared from theSVP26 or Dsvp26 cells expressing either KTR3-3HA or
MNN2-3HA. Microsomal membranes were washed oncewith 0.5 M NaCl in B88 and then 2 times with B88 containing protease inhibitors, and incubated for 30 min at 25oC in

the presence or absence of the purified coat components.
After the incubation, vesicles were separated from thedonor membranes by centrifugation at 10,000 g for 5 min.
The supernatant was further centrifuged at 100,000 g for 1hour to collect the COPII vesicles. The pellets were analyzed by SDS-PAGE and immunoblotting.
RESULTS
Golgi localization of mannosyltransferases Mnn2 andMnn5 depends on Svp26. The most significant phenotypes
of Dsvp26 cells found previously were hyper-mannosylation of N-glycosyl chains on several glycoproteins including invertase and mislocalization of Ktr3 from the Golgi toER (4). As Ktr3 is an O-mannosyltransferase, we examined
if the Golgi localization of N-mannosyltransferases depends on the presence of Svp26, which may cause excess
N-mannosylation. All Golgi N-mannosyltransferases aretype II membrane proteins. We observed the localization oftagged proteins encoded by the modified structural genesthat were integrated in the yeast chromosome of the wildtype or Dsvp26 cells by immunofluorescence microscopy.
In both the Dsvp26 cells and wild-type cells the localizationof Mnn1 and Mnn9 were punctate, as reported previously,
which is a typical pattern of Golgi-localized proteins. The
N-mannosyltransferases Mnn2 and Mnn5 had double ring
signals in most of the Dsvp26 cells as we previously reported for Ktr3, which indicates mislocalization from theGolgi to ER. These results indicate a clear difference in thedependence of N-mannosyltransferases on Svp26 for theirGolgi localization.
Svp26-dependent Golgi proteins Mnn2 and Mnn5 bindto Svp26. We examined the interaction between Mnn2 orMnn5 and Svp26 by co-immunoprecipitation in lysate containing 1% digitonin in order to observe any weak or transient interactions. A significant amount of Svp26 co-immunoprecipitated with Mnn2, although less than theamount that co-immunoprecipitated with Ktr3. No Svp26
was detected in the immunoprecipitate of Mnn1 which does
not depend on Svp26 for its Golgi localization. The amountof Svp26 that was found in the Mnn2 immunoprecipitate
was comparable to amount seen in the immunoprecipitateof Mnn5.
Svp26 stimulates in vitro incorporation of Ktr3 andMnn2 proteins in COPII Vesicles. There are two potential
mechanisms to maintain the localization of a Golgi protein,i.e., to facilitate the arrival of the protein to, or to prevent its
exit from the Golgi. To examine the first possibility, we ex-44

amined if the efficiency to incorporate the mannosyltransferases in COPII vesicles is affected by the presence or absence of Svp26. To test this, we used the in vitro COPII
vesicle formation system with purified coat subunits and an
ER-enriched membrane preparation. When the wild-type
ER-enriched membrane fraction was used, Ktr3 and Mnn2
are released from the ER efficiently by the addition of purified coat subunits. Without the addition of COPII coat components, much less Ktr3 and Mnn2 are released from theER, compared to the positive control, Erv25. The negative
control Sec61 was not released in either condition. Therefore, we conclude that Ktr3 and Mnn2 were efficiently incorporated in the COPII vesicles. In contrast, very little
Ktr3 was released from this Dsvp26 ER fraction by the addition of purified COPII subunits. As Erv25 is released inboth the Dsvp26 and wild-type fractions, the formation andrelease of COPII vesicles occur normally. We basically obtained the similar results as for Mnn2. These results suggestthat the presence of Svp26 strongly stimulates the incorporation of Ktr3 and Mnn2 into COPII vesicles.
Svp26 recognizes the lumenal domain of mannosyltransferase. All Golgi mannosyltransferases identified so
far in S. cerevisiae are type II membrane proteins. Theirpolypeptides can be divided into three parts, i.e. N-terminal
cytoplasmic, short transmembrane and large lumenal domains. The lumenal domain is believed to contribute totheir enzyme activity. We sought to determine which domain is responsible for the differences in Ktr3 or Mnn2
(that depend on Svp26 for Golgi localization) and Mnn1
(that does not need Svp26 to localize in the Golgi) by exchanging their domains. In the wild-type cell, all chimera
proteins were observed in cytoplasmic punctae by immunofluorescent staining. This suggests that they are all localized to Golgi compartments. In the Dsvp26 cells, Mnn1Ktr3 and Mnn1-Mnn2 chimeras are clearly mislocalized tothe ER, while Ktr3-Mnn1 and Mnn2-Mnn1 localized in theGolgi. The localization was rescued by the introduction ofthe wild-type SVP26 by a CEN plasmid. As the ER mislocalization of chimera proteins corresponds to the origin ofthe lumenal domain, this domain is responsible for theSvp26-dependent localization of these proteins strain [5].

Golgi-localization of Kre2 family proteins in the absence of Svp26. Kre2 family comprises of 9 homologous
proteins including Ktr3 and they are thought to catalyze theprotein O-mannosylation in the Golgi. We examined the localization of Kre2 family proteins in the wild type strain
and Dsvp26 strain. We found that Kre2 and Ktr1 were mislocalized to ER in the absence of Svp26. We currently investigating the mechanism by which Svp26 regulates thelocalization of these two proteins.
Conclusions
In this report, in addition to Ktr3, we find that two otherGolgi mannosyltransferases, Mnn2 and Mnn5 are also mislocalized and found in the ER in the absence of Svp26 andthat localization of other mannosyltransferases including
Mnn1 are not affected. Mnn2 and Mnn5 bind to Svp26 invivo as Ktr3 does. Using an in vitro budding assay, the incorporation of Ktr3 and Mnn2 in the COPII vesicles isgreatly 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 andSvp26-independent Mnn1 suggests that the lumenal domainof mannosyltransferases, but not the cytoplasmic or transmembrane domain, is responsible for recognition by Svp26.
These results may be valuable for the future effort to understand the function of the Golgi apparatus in more detail.
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