62NISR Research GRANT
2006 Research Grant

A novel mechanism of intracellular protein localization infilamentous fungi
Hiroyuki HORIUCHI
Department of Biotechnology, The University of Tokyo

Research aims

Methods

Fungal cell walls are dynamic structures and crucial forfungal morphogenesis. Chitin is a homopolymer of Nacetylglucosamine and one of the major cell wall components in many filamentous fungi. To investigate the mechanism of fungal morphogenesis and hyphal tip growth, wehave cloned six chitin synthase-encoding genes from the ascomycete filamentous fungi, Aspergillus nidulans and analyzed their functions. In the course of this study, we havefound two genes, csmA and csmB, encoding chitin synthases that have very unique structures. CsmA and CsmB,
the gene products of csmA and csmB, respectively, consistof a chitin synthase domain, CSD, at their C-termini and amyosin motor-like domain, MMD, at their N-termini (Fig.
1) (1, 2). Myosins are known to be motor proteins thatmove on actin filaments. There are no orthologs of csmAand csmB in the genomes of Saccharomyces cerevisiae,
Schizosaccharomyces pombe, and Candida albicans, butthey are ubiquitous in the genomes of filamentous fungiand dimorphic yeasts of which cell wall chitin contents arerelatively high.
csmA deletion (DA) mutant and csmB deletion (DB) mutant showed growth delay under low osmotic conditions andformed swollen hyphae, called balloons (2, 3). We showedthat both the MMD and the CSD of CsmA were requiredfor its function and that CsmA tagged with HA at its C-terminus localized primarily at hyphal tips and forming septa.
We also showed that a direct interaction between the MMD
and actin was essential for the proper localization of CsmA
(4, 5). These results suggest that CsmA localizes at hyphaltips and forming septa by a novel protein sorting mechanism depending on its MMD. To investigate the mechanism
in detail, we analyzed function of the MMD of CsmB andthe interchangeability of the MMDs between CsmA andCsmB.

We constructed DB mutants that produced a truncatedform of CsmB lacking its MMD under the control of thenative csmB promoter and alcA promoter (alcA(p)), anddesignated them DMB strain and alcA(p)-DMB strain, respectively. The alcA(p) expressed strongly on the medium
containing ethanol or threonine and fructose as carbon
sources. We also constructed a strain that produced thetruncated CsmB tagged with FLAG (DMB-FLAG protein)and determined its intrahyphal localization by indirect immunofluorescence. We made chimera genes encoding theMMD of CsmA and the CSD of CsmB (MACB) or theMMD of CsmB and the CSD of CsmA (MBCA). We constructed strains in which the gene encoding MACB fusion
protein was expressed under the control of csmA promoter
at the argB loci of the DA mutant and the DB mutant anddesignated them MACBDA and MACBDB strains, respectively. Similarly, we constructed MBCADA and MBCADB
strains in which the gene encoding MBCA fusion protein
was expressed under the control of csmA promoter at theargB loci of the DA mutant and the DB mutant, respectively. We also constructed CsmADA and CsmBDB strainsthat expressed the wild-type csmA and csmB at the argBloci of the DA mutant and the DB mutant, respectively. We
constructed a strain that produced FLAG-tagged MACB
protein and determined its intrahyphal localization by indirect immunofluorescence.

Fig. 1.

Strucrures of CsmA and CsmB of Aspergillus nidulans.

Results
1. Function of the MMD of CsmB
The MMD of CsmA contains the consensus sequences ofmyosins, such as P-loop, Switch I, and Switch II, which areessential for the function of myosins. In contrast, those consensus sequences are not found in that of CsmB. To evaluate the importance of the MMD in the function of CsmB,
we constructed the DMB strain and the alcA(p)-DMB strain(see Methods section). The DMB strain grew slowly under
low osmotic conditions and formed balloons, whereas thealcA(p)-DMB strain grew as fast as the wild-type strain andformed balloons less frequently than the DMB strain under
the alcA(p)-inducing conditions. These results suggest thatthe function of CsmB partially depends on its MMD. This
is in contrast to the case of CsmA in which the function ofCsmA was completely dependent on its MMD (3, 5). To
determine the localization of the N-terminally truncated63CsmB in the hyphae, we analyzed the intracellular distribution of DMB-FLAG protein. The wild-type CsmB taggedwith FLAG localized primarily at hyphal tips and forming
septa, whereas DMB-FLAG protein was distributed
throughout hyphae. This indicates that the MMD of CsmB
is important to its proper localization.
2. Interchangeability of the MMDs between CsmA andCsmB
Although deletion of csmA or csmB caused similar defects as described above, overexpression of csmA did notsuppress the defects of the DB mutant and overexpressionof csmB did not suppress those of the DA mutant (2, anddata not shown). These results suggest that CsmA andCsmB have some different functions in growth and morphogenesis. The MMD of CsmB has only 21% amino acid
identity with that of CsmA. To investigate the functional
difference in the MMDs between CsmA and CsmB, weconstructed the MACBDA, MBCADA, MACBDB,MBCADB, CsmDA, and CsmBDB strains (see Methodssection). The CsmADA and CsmBDB strains showed thesame phenotype as the wild-type strain. The MACBDAstrain and the MBCADB strain exhibited the phenotypes
similar to the DA mutant and the DB mutant, respectively,
suggesting that their CSDs are not interchangeable. TheMACBDB strains grew as well as the wild-type strain anddid not form balloons. Although the growth rate of the MBCADA strain was nearly the same as the wild-type strain,
the frequency of balloon formation was not ameliorated tothe level of the wild-type strain. We showed that MACB
tagged with FLAG localized mainly at hyphal tips in theDB mutant.
Conclusion
We demonstrated that the MMD of CsmB was importantto both its proper localization and full function, and that theMMD functions of CsmA and CsmB were partially interchangeable. Since FLAG-tagged MACB localized at hyphaltips, the MMD of CsmA is suggested to have a functionthat is able to target proteins to hyphal tips.
References
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