28[Noda Institute for Scientific Research GRANT]2006 Research Grant
Outline of Research Result

The Pivotal Role of Membrane Traffic in the Growth and Differentiation of Filamentous Fungus, Aspergillus oryzae
Manabu ARIOKA
Department of Biotechnology, The University of Tokyo

Research aims
Secretory and membrane proteins are transported inmembrane-bound vesicles that bud from the donor organelle and fuse with the target membrane. In this process,
SNARE proteins play pivotal roles in providing specificity
and driving force for membrane fusion. In this study, we attempted to characterize SNARE proteins in Aspergillus
oryzae, a filamentous fungus with a prominent ability to secrete proteins. In addition, possible involvement of vacuoles in the developmental process of filamentous fungi
was proposed through in-depth analysis using AoVam3 protein, a vacuolar t-SNARE.
Methods
Amino acid sequences of 24 SNARE proteins in Saccharomyces cerevisiae obtained from the GenBank databasewere used to blast-search the Aspergillus oryzae genome
database. The amino acid sequences of predicted A. oryzae
proteins that showed significant similarity to yeast SNARE
proteins and their open reading frame (ORF) sequenceswere extracted. 5- and/or 3-rapid amplification of cDNAends (RACE) analyses were performed to determine the terminal position of cDNAs. The cDNAs encoding the putative SNAREs were amplified by PCR and used to constructexpression plasmids that contain egfp (enhanced green fluorescent protein) -SNARE fusion genes under the controlof amyB promoter and terminator, and have niaD selection
marker. The plasmids were introduced into A. oryzae NS4
strain, and transformants were obtained. For microscopy,
conidia of A. oryzae strains expressing fusion proteins weregrown for 20 h.

mined by RACE analysis. In addition, the genomic search
identified a sequence that encodes a protein showing weaksimilarity to Use1p. Overall, the search demonstrated thepresence of 21 SNARE-encoding genes in the A. oryzae
genome.
Generation of strains that express fusion proteins of EGFP
with SNAREs
We next generated strains expressing EGFP-SNARE proteins. We chose amyB promoter to drive the expression offusion proteins. All the expression plasmids were constructed by MultiSite Gateway cloning system, using a 5entry clone containing amyB promoter, a center entry clone
containing egfp, 3 entry clones containing cDNAs encoding SNAREs, and a destination vector containing amyB terminator and niaD selection marker. The resultant plasmids
were introduced into A. oryzae NS4 strain and at least fourtransformants were obtained.

Results

Four putative SNAREs that localize to ER
A. oryzae has a set of SNARE orthologs in the transportof ER to Golgi; AoUfe1, AoSec20, AoUse1, and AoSec22.
The fluorescence in the strains expressing EGFP-fused
AoUfe1, AoSec20, AoUse1, and AoSec22 was primarilyobserved at ring-like structures, while a part of the fluorescence was observed at tubular or diffused structures in thecytoplasm. Staining with DAPI demonstrated that the fusion proteins were localized in the ring-like structures surrounding nuclei, suggesting that they are perinuclear ER. Inaddition, EGFP-AoSec20 and -AoUse1 resided in linear
compartments near the plasma membrane (PM) and theseptum, and were not stained with ER-Tracker. This result
may reflect the presence of peripheral ER in filamentous
fungi, and the preferential localization of AoSec20 andAoUse1 to this compartment.

Identification of 21 genes encoding putative SNARE proteins in A. oryzae
We first searched the A. oryzae genome database for annotated open reading frames (ORFs) that encode proteinsshowing high similarity to SNARE proteins of S.
cerevisiae. The search demonstrated the presence of 20 annotated ORFs except for Use1p ortholog. For some genes,5 and/or 3 ends of the cDNAs were experimentally deter-Six putative SNAREs that localize to the Golgi apparatus
In strains expressing the either one of six EGFP-SNAREfusion proteins (EGFP-AoSed5, -AoBos1, -AoGos1,-AoBet1, -AoSft1, and -AoVti1), EGFP fluorescenceshowed similar distributions as punctate structures often
found at the apical regions, or round structures (presumablyvacuoles) in the proximal regions. In strains expressing
EGFP-AoYkt6, EGFP fluorescence was present in the en-29tire cytoplasm and not restricted to any compartments; thisseemed be due to mislocalization.
Four putative SNAREs that localize to the plasma membrane
The fusion proteins of AoSso1 and AoSso2 with EGFP
localized to the plasma membrane and the septum, suggesting that these two putative SNAREs act in exocytosis. Also,AoSnc1 and AoNyv1 both localized to the plasma membrane, but the fluorescence of EGFP-AoNyv1 at the plasma
membrane was far weaker than that of EGFP-AoSnc1.
Therefore, AoNyv1 seemed to function elsewhere. In all
strains in which EGFP fluorescence was observed at theplasma membrane, the fluorescence evenly covered the cellsurface except that it tended to be weak at hyphal tips, andno particular tip-high gradient of the fluorescence was observed. In strains expressing EGFP-AoSec9, the fluorescence occupied the entire cytoplasm, as in the strains expressing EGFP-AoYkt6. We speculated that the fusion with
EGFP might have inhibited the anchorage to the plasma
membrane of AoSec9.
Three putative SNAREs that localize to vacuolar membrane
Vacuolar localization of AoVam3 in A. oryzae has beenreported. As AoVam3, EGFP-AoVti1 and -AoVam7 localized to the membrane of vacuoles that were stained byCMAC. EGFP-AoNyv1 also localized to the vacuolar
membrane, suggesting that AoNyv1 has dual roles as exocytic and vacuolar R-SNAREs.
Putative SNAREs that localize to endosomes
In filamentous fungi, early endosomes are thought to be
the compartments that show rapid (5 m m/sec) movement
along the hyphal axis, while late endosomes are thought tobe static, adjacent to vacuoles and can be stained by FM464. The fusion proteins of five SNARE orthologs, AoTlg1,AoTlg2, AoVti1, AoSyn8, and AoSnc1 localized to mobile
particulate compartments. The compartments were stainedby FM4-64. The fusion proteins of five SNAREs, AoTlg2,AoVti1, AoNyv1, AoVam7, and AoSyn8 localized to staticcompartments. The former and latter structures were suggested to be early and late endosomes, respectively.
Putative SNAREs that localize to the septum
Some of the fusion proteins of SNAREs localized at/near
the septum. These include three groups of SNAREs; theplasma membrane SNAREs AoSso1, AoSso2, and AoSnc1;
ER SNAREs AoSec20 and AoUse1; and endosomal/vacuolar SNAREs AoTlg1, AoVti1, and AoNyv1. EGFP fluorescence of the fusion proteins of the plasma membrane
SNAREs most likely reflects the septal membrane. Instrains expressing the fusion proteins of ER SNAREs,
EGFP fluorescence seemed to be present as doublet on bothsides of the septum of two adjacent compartments.
A novel role of vacuoles in filamentous fungi
Next we analyzed the localization of AoVam3-EGFP in

more detail. In young hyphae AoVam3-EGFP localized on
the vacuolar membrane, while in the old hyphae cultured
for several days the EGFP fluorescence was distributed into
the vacuolar lumen. Since the EGFP moiety of the fusion
protein resides on the cytoplasmic side of the vacuoles, this
topological change of EGFP fluorescence imply that thevacuolar membrane invaginated into the lumen, presumably
by budding into the luminal side of the vacuole. To examine
the possibility that in the old hyphae other cytoplasmic contents are similarly transported into the vacuole, we observed the localization of nuclei, mitochondria, and peroxisome; these organelles were visualized by the expression ofEGFP-fusion proteins that specifically localize to the corresponding organelles. As expected, the EGFP fluorescence
was exclusively observed in each organelle in young hyphae. In contrast, the fluorescence was also observed in thevacuoles in old hyphae. Moreover, this shift in localization
did not occur in the strain deleted for Aoatg8, a gene essential for autophagy. Taken together, these results suggest thatvacuoles in the old hyphae of filamentous fungi take up anddegrade cytoplasmic organelles via autophagy, presumably
for the purpose of recycling cellular components.
Conclusion
In summary, the localization and possibly function of theER, Golgi, and endosomal SNAREs seem to be largely conserved between S. cerevisiae and A. oryzae. It is thus likelythat the fluorescence of EGFP-SNARE reflects the spatial
distribution of each organelle where the SNARE protein localizes. Some SNAREs localized to more than one cellularcompartments. Intriguingly, the analysis using AoVam3
demonstrated that the fungal vacuoles in the old hyphaetake up various organelles. Since this process was dependent on the presence of the autophagy gene, it is suggested
that the vacuoles in filamentous fungi play pivotal roles indegrading unnecessary intracellular organelles in the oldhyphae and recycling cellular components to young, actively-growing hyphae.
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