Inactivation of the KlPMR1 gene of Kluyveromyces lactis results in defective cell-wall morphogenesis

Abstract

The P-type Ca2+-ATPases are the transporters responsible for calcium homeostasis in the cell compartments of eukaryotes. The KlPMR7 gene of KIuyveromyces lactis encodes a P-type Ca*+-ATPase, which is functionally and structurally homologous to Pmrl p of Saccharomyces cerevisiae, the calcium pump localized in the Golgi membranes. In this work, a novel involvement of KlPmrlp in cell-wall morphogenesis of K, lactis is reported. KlpmrIA cells exhibited the loss of outer-chain extension in the glycosylation of secreted proteins. The absence of KlPmrlp resulted in the accumulation of round, large cells with an abnormally thick cell wall, as revealed by transmission electron microscopy. The deletant strain also showed a delocalized deposition of chitin in the lateral cell wall accompanied by an unbalanced ratio of insoluble to soluble glucans. These morphological defects were accompanied by the presence of irregularly shaped nuclei and by a DNA content greater than 2n. Addition of I 0 mM Ca2+ to the medium of the KlpmrIA strain reversed the chitin-deposition impairment, recovered the alteration to the glucan ratio and restored a normal thickness of the cell wall. The mutant cells resumed wildtype size, shape and nuclear morphology but the DNA content indicated the persistence of defects in the co-ordination between DNA replication and cell division. The glycosylation defects were completely unaffected by the calcium supplement. These results indicate that calcium homeostasis controlled by KlPmrlp plays an important role in the cell-wall morphogenesis of K. lactis.

Functional Expression in Yeast of the Human Secretory Pathway Ca2+, Mn2+-ATPase Defective in Hailey-Hailey Disease

“…Nonsense and missense mutations inactivating one allele of hSPCA1 are found in patients with Hailey-Hailey disease (MIM 16960), whose symptoms involve a loss of ketatinocyte cohesion (22,23). This defect is reminiscent of improper protein glycosylation, sorting, and cell wall morphogenesis in pmr1-null mutants (4,24,25). In this study, we present direct biochemical evidence that hSPCA1 is a bona fide member of the secretory pathway Ca 2ϩ -ATPases.…”

Schizosaccharomyces pombe Pmr1p Is Essential for Cell Wall Integrity and Is Required for Polarized Cell Growth and Cytokinesis

“…A phenotypic relationship between loss of Pmr1p function and an abnormal cell wall morphology was first reported in Kluyveromyces lactis (67). Klpmr1⌬ cells revealed several defective phenotypes, including incomplete protein glycosylation, aberrant chitin deposition, and a thickened cell wall with an unbalanced ratio of insoluble to soluble glucans.…”

“…Cell wall weakness thus can be attributed to the dramatic changes in the composition of the mutant cell wall. It should be noted that deletion of S. cerevisiae PMR1 does not result in a detectable change in cell wall composition, in spite of the glycosylation defects (67).…”

KlMID1, a relevant key player between endoplasmic reticulum homeostasis and mitochondrial dysfunction in Kluyveromyces lactis

“…Exponential-phase cells grown in SD minimal medium were harvested, washed with water and then incubated with the vital dye 2-(4-dimethylaminostyryl)-N-methylpyridinium iodide (DASPMI; Invitrogen) by the method of Guthrie & Fink (2002). Chitin staining was performed by the method of Uccelletti et al (1999). Epifluorescence microscopy was carried out with a Zeiss AxioVert 25 microscope fitted with a 6100 immersion objective and a standard filter set.…”