Subcellular localization, abundance and stability of chitin synthetases 1 and 2 from Saccharomyces cerevisiae

Abstract

The existence of more than one chitin synthetase in fungal cells poses the question of whether these enzymes have similar or different localization. The subcellular distribution of chitin synthetases 1 and 2 (Chsl and Chs2) was determined in cell-free extracts of Saccharomyces cerevisiae fractionated by sucrose density gradient sedimentation. Chsl was examined in two strains : ATCC 26109, a wild-type strain, and D3C (MATE ura3-52). Chs2 was investigated in a strain (D3B) freed of Chsl by gene disruption (MATa his4 ura3-52 chsl::URA3). A prolonged, strong centrifugation (20 h a t 265000 g) was necessary to cleanly resolve two major populations of chitin synthetase particles : chitosomes (a population of microvesicles of low buoyant density, d = 1-15 g ml-I) and plasma membrane (a population of vesicles of high buoyant density, d = 1.21 g ml-I). Chsl and Chs2 were both present in chitosomes and plasma membrane, but the relative distribution of each chitin synthetase in these two membranous populations varied. Chs2 was much less abundant than Chsl and required Co2+ rather than Mg2+ as a cofactor. A salient finding was the high sensitivity of chitosomal Chs2 to high centrifugal forces. The subcellular distribution of 1,3-P-glucan synthetase was the same in the three strains studied, i.e. unaffected by the presence or absence of Chsl. Culture conditions affected the profiles of chitin and glucan synthetases : the relative abundance of Chsl in chitosomes or plasma membrane was quite different in cells grown on two different media but the buoyant density was not affected; in contrast, there was shift in the buoyant density of the two peaks of 1,3-P-glucan synthetase. We concluded that the subcellular localization of Chsl and Chs2 remains the same despite genetic and other differences in the properties of these enzymes. We confirmed that 1,3-P-glucan synthetase and chitin synthetase exhibit a partially different subcellular distribution -an indication that these two enzymes are mobilized through different secretory pathways.

Spitzenkörper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of Neurospora crassa

“…Our findings on the localization of CHS-3 and CHS-6 in N. crassa, plus previous studies on the three CHS from S. cerevisiae, Chs1p, Chs2p (31), and Chs3p (17), show that they all can be detected in chitosomes. This raises the question as to whether these different CHS belong to one multienzyme complex transported within the same population of chitosomes or Three observations argue against the involvement of the conventional ER-to-Golgi body secretory pathway in the intracellular traffic of CHS but in favor of a new model for an unconventional, separate, CHS secretory pathway: (i) the inability of BFA to diminish the fluorescence reaching the Spk; (ii) the lack of colocalization between Golgi body equivalents and ER membranes and CHS-GFP-stained membranes; and (iii) results from previous studies showing that the ER microvesicles and chitosomes of N. crassa (30) belong to different populations that can be separated by high-performance ultracentrifugation (31,56).…”

“…This raises the question as to whether these different CHS belong to one multienzyme complex transported within the same population of chitosomes or Three observations argue against the involvement of the conventional ER-to-Golgi body secretory pathway in the intracellular traffic of CHS but in favor of a new model for an unconventional, separate, CHS secretory pathway: (i) the inability of BFA to diminish the fluorescence reaching the Spk; (ii) the lack of colocalization between Golgi body equivalents and ER membranes and CHS-GFP-stained membranes; and (iii) results from previous studies showing that the ER microvesicles and chitosomes of N. crassa (30) belong to different populations that can be separated by high-performance ultracentrifugation (31,56). Both the gradual decrease in size of the GFP-labeled globular vacuoles from the subapical region of the hyphae to the apex and the findings on the intracellular movement point to a progressive dispersion of the CHS-containing structures as they move towards the growing tip.…”

Cell Wall Assembly in Saccharomyces cerevisiae

“…The third CHS gene, CHS1, shows an expression pattern similar to that of CHS3, peaking in early G 1 , while the Chs1 level remains constant during the cell cycle (215,315). Chs1 shows the same dual subcellular localization as Chs3, with most of the protein found in chitosomes and a minor fraction found at the plasma membrane (147,315). Since Chs1 and Chs3 both populate chitosome vesicles, they are thought to follow the same route.…”

The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenkörper in Neurospora crassa

“…S4A and B), corresponding to 1.155 and 1.158 g/ml. This indicated that PMA-1 travels in high-density vesicles and is found at the PM (34,35).…”