Purification and characterization of cell wall lytic enzyme released by mating gametes of <i>Chlamydomonas reinhardtii</i>

Matsuda, Yoshihisa; Yamasaki, Akira; Saito, Tatsuaki; Yamaguchi, Takahiro

doi:10.1016/0014-5793(84)80098-8

Cited by 38 publications

(31 citation statements)

References 23 publications

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“…Nonetheless, granular structures are visible in some layers of the Chlamydomonas cell wall (64,65). Insights into the identity of the Golgi aggregates could be gained by correlating their abundance with conditions that increase cell wall secretion, such as synchronized growth-phase cultures and recovery from treatment with the autolysin enzyme, which removes the cell wall (66,67).…”

Section: Resultsmentioning

confidence: 99%

In situ structural analysis of Golgi intracisternal protein arrays

Engel

Schaffer

Albert

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

106

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We acquired molecular-resolution structures of the Golgi within its native cellular environment. Vitreous Chlamydomonas cells were thinned by cryo-focused ion beam milling and then visualized by cryo-electron tomography. These tomograms revealed structures within the Golgi cisternae that have not been seen before. Narrow trans-Golgi lumina were spanned by asymmetric membrane-associated protein arrays that had ∼6-nm lateral periodicity. Subtomogram averaging showed that the arrays may determine the narrow central spacing of the trans-Golgi cisternae through zipper-like interactions, thereby forcing cargo to the trans-Golgi periphery. Additionally, we observed dense granular aggregates within cisternae and intracisternal filament bundles associated with trans-Golgi buds. These native in situ structures provide new molecular insights into Golgi architecture and function.focused ion beam | cryo-electron tomography | Chlamydomonas | Golgi | glycosyltransferase C ryo-electron tomography (cryo-ET) provides the unique ability to visualize macromolecules and supramolecular structures within frozen hydrated cells (1-4). Biological material is immobilized in vitreous ice, preserving cellular structures in a near-native state. Compression-free thinning of these frozen samples by cryofocused ion beam (cryo-FIB) milling offers unparalleled access to the cellular interior (5-7). The recent combination of cryo-FIB with the improved image quality of direct detection cameras has opened new frontiers for in situ structural biology, enabling the study of how molecular complexes establish cellular architecture.The relationship between Golgi structure and function has been intensely debated since the first electron microscopy observations of this alluring organelle (8,9). Over the last two decades, electron tomography of plastic sections has been applied extensively to characterize Golgi morphology within animals, plants, and single-celled organisms, including yeast and algae (10-17). Three-dimensional views of fenestrated, interconnected cisterna stacks interacting with a constellation of coated vesicles led to revised models of how Golgi structure directs cargo sorting through the organelle (18-20). However, these tomographic studies were restricted to descriptions of membrane architecture and, in the best cases, the classification of membrane coats, due to the resolution limitations imposed by conventional sample preparation, involving dehydration, plastic embedding, and staining with heavy-metal contrasting agents. To date, cryo-ET studies of the Golgi have been extremely limited (2,(21)(22)(23).In this study, we used cryo-FIB of vitreous Chlamydomonas cells followed by cryo-ET to image the native molecular landscape of the Golgi with unprecedented resolution and sample integrity. Our tomograms revealed new structures within the Golgi cisternae, including ordered membrane-associated protein arrays, dark granular aggregates, and bundles of filaments near the trans-Golgi coated buds. Results and DiscussionTrans-Golgi Intracisternal...

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Section: Resultsmentioning

confidence: 99%

In situ structural analysis of Golgi intracisternal protein arrays

Engel

Schaffer

Albert

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

106

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“…It was inhibited by several serine protease inhibitors and not by other types of protease inhibitors including phenanthroline, a metalloprotease inhibitor that completely inhibits lysin (14,16).…”

Section: Discussionmentioning

confidence: 99%

Regulated secretion of a serine protease that activates an extracellular matrix-degrading metalloprotease during fertilization in Chlamydomonas.

Snell

Eskue

Buchanan

1989

The Journal of Cell Biology

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Abstract. During fertilization in the biflagellated alga, Chlamydomonas reinhardtii, gametes of opposite mating types adhere to each other via agglutinin molecules located on their flagellar surfaces, generating a sexual signal that induces several cellular responses including cell wall release. This cell contact-generated signal is mediated by cAMP and release of the wall, which is devoid of cellulose and contains several hydroxyproline-rich glycoproteins, is due to the activation of a metalloprotease, lysin. Although we originally assumed that lysin would be stored intracellulady in a compartment structurally separate from its substrate, recently we showed that lysin is stored in the periplasm as an inactive, higher relative molecular mass precursor, prolysin (Buchanan, M. J., S. H.

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“…This question was first approached by inactivation of GLE with either EDTA or by heat treatment (Matsuda et al, 1984(Matsuda et al, , 1987. Vegetative cells treated with autolysin for 2 h either without EDTA or with 1 or 2 mM EDTA were compared (Fig.…”

Section: Accumulation Of Gas Mrnas After Cell Wall Removalmentioning

confidence: 99%

“…It was shown previously that the activity of purified GLE is lost completely by a treatment at 50°C for 5 min (Matsuda et al, 1984). The autolysin suspension was incubated at 50°C for 10 min prior to addition to vegetative cells.…”

Section: Accumulation Of Gas Mrnas After Cell Wall Removalmentioning

confidence: 99%

Mating-Induced Shedding of Cell Walls, Removal of Walls from Vegetative Cells, and Osmotic Stress Induce Presumed Cell Wall Genes in Chlamydomonas

Hoffmann

Beck

2005

Plant Physiology

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The first step in sexual differentiation of the unicellular green alga Chlamydomonas reinhardtii is the formation of gametes. Three genes, GAS28, GAS30, and GAS31, encoding Hyp-rich glycoproteins that presumably are cell wall constituents, are expressed in the late phase of gametogenesis. These genes, in addition, are activated by zygote formation and cell wall removal and by the application of osmotic stress. The induction by zygote formation could be traced to cell wall shedding prior to gamete fusion since it was seen in mutants defective in cell fusion. However, it was absent in mutants defective in the initial steps of mating, i.e. in flagellar agglutination and in accumulation of adenosine 3#,5#-cyclic monophosphate in response to this agglutination. Induction of the three GAS genes was also observed when cultures were exposed to hypoosmotic or hyperosmotic stress. To address the question whether the induction seen upon cell wall removal from both gametes and vegetative cells was elicited by osmotic stress, cell wall removal was performed under isosmotic conditions. Also under such conditions an activation of the genes was observed, suggesting that the signaling pathway(s) is (are) activated by wall removal itself.

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Purification and characterization of cell wall lytic enzyme released by mating gametes of Chlamydomonas reinhardtii

Cited by 38 publications

References 23 publications

In situ structural analysis of Golgi intracisternal protein arrays

In situ structural analysis of Golgi intracisternal protein arrays

Regulated secretion of a serine protease that activates an extracellular matrix-degrading metalloprotease during fertilization in Chlamydomonas.

Mating-Induced Shedding of Cell Walls, Removal of Walls from Vegetative Cells, and Osmotic Stress Induce Presumed Cell Wall Genes in Chlamydomonas

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