Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

Komine, Yutaka; Ll, Eggink; Park, H; Jk, Hoober

doi:10.1007/s004250050695

Cited by 71 publications

(49 citation statements)

References 35 publications

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“…The ars76 mutant cells also lack vacuoles and polyphosphate bodies (Figure 4). Interestingly, as also observed by Komine et al (2000), we noted Golgi stacks positioned near the acidocalcisome in wild-type 21 gr, suggesting a possible function for these organelles in protein trafficking ( Figure 5; Supplemental Figure 3). We observed this in the C 6-2 rescued strain ( Figure 4A); however, since there are no identifiable acidocalcisomes in the ars76 mutant and most cells already showed deformations, this phenomenon could not be observed in ars76 ( Figure 4B).…”

Section: Structure and Function Of Vtc1supporting

confidence: 80%

“…The mutant strain also showed a relatively normal decline in the level of mRNA for the low affinity sulfate transporter, SULTR1, during S deprivation. Congruent with the RT-qPCR data, levels of transporter proteins were also significantly lower in ars76 than in wild-type 21 gr Region marked with an arrow in Figure 3B but at 80003 magnification, showing vesicles potentially leaving Golgi stocks and fusing with vacuoles as described by Komine et al (2000). The positions of the vacuoles are interior to the apical regions of chloroplast (see Figure 3B).…”

Section: Expression Of S-deprivation-responsive Genes In the Ars76 Musupporting

confidence: 55%

“…For example, recycling macromolecules in the vacuole or developing acidocalcisome may be critical for acquiring building blocks needed for the synthesis of new sets of proteins, lipids, and other compounds as the cells acclimate to new conditions. However, it is also possible that as the cells experience nutrient limitation the forming vacuoles might, as suggested by Komine et al (2000), be directly involved in packaging material for transport to various cellular locations, including the periplasmic space.…”

Section: Discussionmentioning

confidence: 99%

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Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation

2014

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Forward genetics was used to isolate Chlamydomonas reinhardtii mutants with altered abilities to acclimate to sulfur (S) deficiency. The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex. The ars76 mutant cannot accumulate arylsulfatase protein or mRNA and shows marked alterations in levels of many transcripts encoded by genes induced during S deprivation. The mutant also shows little acidocalcisome formation compared with wild-type, S-deprived cells and dies more rapidly than wild-type cells following exposure to S-, phosphorus-, or nitrogen (N)-deficient conditions. Furthermore, the mutant does not accumulate periplasmic l-amino acid oxidase during N deprivation. Introduction of the VTC1 gene specifically complements the ars76 phenotypes, suggesting that normal acidocalcisome formation in cells deprived of S requires VTC1. Our data also indicate that a deficiency in acidocalcisome function impacts trafficking of periplasmic proteins, which can then feed back on the transcription of the genes encoding these proteins. These results and the reported function of vacuoles in degradation processes suggest a major role of the acidocalcisome in reshaping the cell during acclimation to changing environmental conditions.

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Section: Structure and Function Of Vtc1supporting

confidence: 80%

Section: Expression Of S-deprivation-responsive Genes In the Ars76 Musupporting

confidence: 55%

Section: Discussionmentioning

confidence: 99%

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Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation

2014

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“…These vacuoles, called acidocalcisomes, are acidified compartments that store polyphosphate complexed with high concentrations of cations, including calcium, iron, zinc, and copper (46)(47)(48). Thus, an alternative candidate for the trans-Golgi filaments is polyphosphate, the major nonorganic component of acidocalcisomes (49)(50)(51). These filament bundles could also contain the enzyme polyphosphate kinase 2, which forms actin-like fibers concurrent with polyphosphate synthesis (52).…”

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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“…However, such a process has not yet been described for Chlamydomonas, although treatment of this alga with rapamycin resulted in a pronounced increase in the size of vacuoles, which resembled an autophagy-like process (Crespo et al, 2005). Interactions between chloroplasts and vacuoles have been observed (Park et al, 1999;Komine et al, 2000). In particular, a pathway for degradation through transfer of proteins from the chloroplast to vacuoles in Chlamydomonas has been suggested, based on the finding that several chloroplast proteins were found in dense granules in cytoplasmic vacuoles.…”

Section: Alb32 Is Essential For Cell Survival In Chlamydomonasmentioning

confidence: 99%

One of Two Alb3 Proteins Is Essential for the Assembly of the Photosystems and for Cell Survival in Chlamydomonas

et al. 2006

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Proteins of the YidC/Oxa1p/ALB3 family play an important role in inserting proteins into membranes of mitochondria, bacteria, and chloroplasts. In Chlamydomonas reinhardtii, one member of this family, Albino3.1 (Alb3.1), was previously shown to be mainly involved in the assembly of the light-harvesting complex. Here, we show that a second member, Alb3.2, is located in the thylakoid membrane, where it is associated with large molecular weight complexes. Coimmunoprecipitation experiments indicate that Alb3.2 interacts with Alb3.1 and the reaction center polypeptides of photosystem I and II as well as with VIPP1, which is involved in thylakoid formation. Moreover, depletion of Alb3.2 by RNA interference to 25 to 40% of wild-type levels leads to a reduction in photosystems I and II, indicating that the level of Alb3.2 is limiting for the assembly and/or maintenance of these complexes in the thylakoid membrane. Although the levels of several photosynthetic proteins are reduced under these conditions, other proteins are overproduced, such as VIPP1 and the chloroplast chaperone pair Hsp70/ Cdj2. These changes are accompanied by a large increase in vacuolar size and, after a prolonged period, by cell death. We conclude that Alb3.2 is required directly or indirectly, through its impact on thylakoid protein biogenesis, for cell survival.

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Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components

Cited by 71 publications

References 35 publications

Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation

Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation

In situ structural analysis of Golgi intracisternal protein arrays

One of Two Alb3 Proteins Is Essential for the Assembly of the Photosystems and for Cell Survival in Chlamydomonas

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