A Novel Factor Essential for Unconventional Secretion of Chitinase Cts1

Abstract

Subcellular targeting of proteins is essential to orchestrate cytokinesis in eukaryotic cells. During cell division of Ustilago maydis , for example, chitinases must be specifically targeted to the fragmentation zone at the site of cell division to degrade remnant chitin and thus separate mother and daughter cells. Chitinase Cts1 is exported to this location via an unconventional secretion pathway putatively operating in a lock-type manner. The underlying mechanism is largely unexplored. Here, we applied a forward genetic screen based on UV mutagenesis to identify components essential for Cts1 export. The screen revealed a novel factor termed Jps1 lacking known protein domains. Deletion of the corresponding gene confirmed its essential role for Cts1 secretion. Localization studies demonstrated that Jps1 colocalizes with Cts1 in the fragmentation zone of dividing yeast cells. While loss of Jps1 leads to exclusion of Cts1 from the fragmentation zone and strongly reduced unconventional secretion, deletion of the chitinase does not disturb Jps1 localization. Yeast-two hybrid experiments indicate that the two proteins might interact. In essence, we identified a novel component of unconventional secretion that functions in the fragmentation zone to enable export of Cts1. We hypothesize that Jps1 acts as an anchoring factor for Cts1.

Establishing regulated unconventional secretion for production of heterologous proteins

“…Microscopic analysis revealed that, as expected, the regulated strains grew yeastlike without any morphological phenotype both in glucose and in arabinose-containing media. However, in contrast to previous localization studies [24], Jps1-Gfp mainly formed intracellular aggregates (about 80%) during all stages of cytokinesis, with only a minor population of about 3% showing the expected localization in the fragmentation zone in late cytokinesis when transcription was induced by arabinose ( Fig. 2B).…”

“…The presence of the potential anchoring factor Jps1 is essential for unconventional Cts1 secretion via the fragmentation zone (Fig. 1B, C) [24]. This mechanistic insight might provide the unique possibility of using Jps1 as a regulator for unconventional protein secretion and thus, to establish a first inducible system.…”

“…Oligonucleotides applied for cloning are listed in Table 1. The generation of pUMa3329_Δupp1_Pcrg-eGfp-Tnos-natR, pUMa2113_pRabX1-Poma_gus-SHH-cts1, pUMa2240_Ip_Poma-his-αGfpllama-ha-Cts1-CbxR and pUMa2775_um03776D_hyg had been previously described [14,22,24,25] but often used in differing strain backgrounds in the present study (for references see Table 2). For generation of pUMa4234_Δupp1_Pcrg-jps1-eGfp-Tnos-natR and pUMa4235_Δupp1_Pcrg-jps1-Tnos-natR, jps1-gfp or jps1 were amplified and inserted into a upp1 insertion vector.…”

“…Recently, we obtained first insights into the cellular mechanism of unconventional secretion [23][24][25]. During cytokinesis of yeast cells, a primary septum is formed at the mother cell side, followed by a secondary septum at the daughter cell side, delimiting a so-called fragmentation zone ( Fig.…”

Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

“…Recently, we obtained the first insights into the cellular mechanism of unconventional secretion [ 29 , 30 , 31 ]. During cytokinesis of yeast cells, a primary septum is formed at the mother cell side, followed by a secondary septum at the daughter cell side, delimiting a so-called fragmentation zone ( Figure 1 A) [ 32 ].…”