J. Microbiol. Biotechnol. (2011), 21(4), 412–420 doi: 10.4014/jmb.1009.08042 First published online 25 January 2011 Evidence of an Alternative Route of Cellobiase Secretion in the Presence of Brefeldin A in the Filamentous Fungus Termitomyces clypeatus Banik, Samudra Prosad 2 , Swagata Pal 1 , Sudeshna Chowdhury 1 , Shakuntala Ghorai 2 , and Suman Khowala 1 * Drug Development and Biotechnology, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata-700032, India Department of Microbiology, Maulana Azad College, 8 Rafi Ahmed Kidwai Road, Kolkata-700013, India Received: September 27, 2010 / Revised: January 10, 2011 / Accepted: January 11, 2011 Secretion of cellobiase occurred in a brefeldin A (BFA) uninhibited manner in the filamentous fungus Termitomyces clypeatus. Fluorescence confocal microscopy revealed that application of the drug at a concentration of 50 µg/ml caused arrest of Spitzenkorper assembly at the hyphal tip. This resulted in greater than 30% inhibition of total protein secretion in the culture medium. However, the cellobiase titer increased by 17%, and an additional 13% was localized in the vacuolar fraction en route secretion. The secretory vacuoles formed in the presence of the drug were also found to be bigger (68 nm) than those in the control cultures (40 nm). The enzyme secreted in the presence and absence of BFA revealed a single activity band in both cases in native PAGE and had similar molecular masses (approx. 120 kDa) in SDS-PAGE. The BFA enzyme retained 72% of native glycosylation. It also exhibited a higher stability and retained 98% activity at 50 o C, 93.3% activity at pH 9, 63.64% activity in the presence of 1 M guanidium hydrochloride, and 50% activity at a glucose concentration of 10 mg/ml in comparison to 68% activity, 75% activity, 36% activity, and 19% activity for the control enzyme, respectively. The observations collectively aimed at the operation of an alternative secretory pathway, distinct from the target of brefeldin A, which bypassed the Golgi apparatus, but still was able to deliver the cargo to the vacuoles for secretion. This can be utilized in selectively enhancing the yield and stability of glycosidases for a successful industrial recipe. Keywords: Termitomyces clypeatus, Spitzenkorper, brefeldin A, cellobiase, filamentous fungi, fungal secretory pathway Filamentous fungi have been considered as excellent production machineries of industrial enzymes for ages owing to their natural ability to secrete large titers of these enzymes into the growth medium. They are also preferentially chosen over plant, insect, and mammalian cell lines as expression hosts of heterologous proteins in terms of both quantity and cost of production [41]. Increased production is achieved by rigorous strain improvement programmes through various molecular genetic means [23], like introduction of a large number of gene copies, the use of strong fungal transcription-control regions and efficient secretion signals [39], construction and use of protease- deficient host strains [40], and development of an optimal production medium. However, our limited knowledge about the secretory apparatus of these organisms remains a serious bottleneck in achieving substantial improvement of the secretory titer. The secretion pathway in fungi differs from those of yeast and higher eukaryotes [7]. Filamentous fungi possess a polarized secretory apparatus [2, 10, 13]. Protein secretion occurs through the apical or subapical regions of the hyphae through a structure unique to the filamentous fungi, the “Spitzenkorper (Spk)” [11]. This is an aggregate of vesicles postulated to act as a center for vesicle supply and to direct their transport to the plasma membrane. Brefeldin A (BFA), a fungal macrocyclic lactone, is a potent inhibitor of protein trafficking through the endomembrane system of mammalian cells [20]. The target of BFA is a subset of Sec-7 like GTP exchange factors that catalyze the activation of a small GTPase called Arf-1p [14]. Arf-1p is responsible for recruitment of coatomer proteins (COP I), as well as clathrin via adaptor complex AP-1 to membranes, resulting in the formation of transport vesicles. Arf-1p and BFA sensitive guanidine exchange factors (GEFs) are localized to the Golgi apparatus of mammalian and yeast cells. Until now, little is known about the subcellular effects of BFA in filamentous fungi [3, 27, 30, 33]. The present study focused on the effects of BFA on the filamentous fungus Termitomyces clypeatus, where, unlike other fungi, secretion of proteins is under the regulation *Corresponding author Phone: +91-33-24995813; Fax: +91-33-24735197, 91-33-24730284; E-mail: sumankhowala@iicb.res.in, sumankhowala@yahoo.com