Membrane-bound and free polysomes in Dictyostelium discoideum: I. Isolation and developmental effects on size and distribution

doi:10.1016/0012-1606(81)90440-1

Developmental Biology

Volume 82, Issue 1, February 1981, Pages 180-185

https://doi.org/10.1016/0012-1606(81)90440-1 Get rights and content

Abstract

Using a simple one-step procedure undegraded membrane-bound polysomes relatively uncontaminated with free polysomes were purified from the cellular slime mold, Dictyostelium discoideum. In axenically grown vegetative cells 17–18% of the total ribosomes are associated with membranes. This value drops slightly during the aggregation stage of development, increases to vegetative levels in pseudoplasmodia, and again drops slightly during fruiting body construction. Within 15 min after development is initiated by the removal of nutrients, both the total and membrane-bound polysomes decrease in size from an average of 9–11 ribosomes per mRNA to an average of 7 or 8 ribosomes per mRNA. The percentage of total ribosomes found as monosomes increases from 10 to 55%. Both total and membrane-bound polysomes remain at this reduced size throughout development.

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Translational control of protein synthesis during early stages of differentiation of the slime mold Dictyostelium discoideum
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Cited by (13)

Ribosomal protein gene expression is cell type specific during development in Dictyostelium discoideum
1999, Differentiation
Starvation for amino acids initiates the developmental cycle in the cellular slime mold, Dictyostelium discoideum. Upon starvation one of the earliest developmental events is the selective loss of the ribosomal protein mRNAs from polysomes. This loss depends upon sequences in the 5′ non-translated leader of the ribosomal protein (r-protein) mRNAs. Here evidence is presented which indicates that those cells which will become prestalk cells express the ribosomal protein genes during development under starvation conditions. Cells which enter the prespore pathway shut off r-protein synthesis. The promoter and 5′ non–translated leader sequences from two ribosomal protein genes, the rp-L11 and the rp–S9 genes, are fused to the Escherichia coli β-galactosidase reporter gene. While β-galactosidase enzyme activity is detected in situ in most growing cells, by 15 h of development β-galactosidase enzyme activity is largely lost from the prespore cells although strong β-galactosidase enzyme activity is present in the prestalk cells. These observations suggest the possibility that the ribosomal protein mRNAs are excluded from polysomes in a cell-type-specific manner.
Tales of poly(A): a review
1990, Gene
Until recently, evidence to support a translational role for the 3′-poly(A) tract of eukaryotic mRNAs has been mostly indirect, including: a correlation between the adenylation status of individual mRNAs and their translatability in vivo or in vitro, the demonstration that exogenously added poly(A) is a potent competitive inhibitor of the translation of poly(A)⁺mRNA, but not poly(A)⁻mRNAs in vitro, and a correlation between the abundance and stability of poly(A)-binding proteins (PABPs) and the rate of translational initiation in vivo. However, more recent studies demonstrate directly that poly(A)⁺mRNAs can initiate translation more efficiently than poly(A)⁻mRNAs, and indicate that this effect is: (i) targeted to the formation of 80S initiation complexes, and (ii) likely to be mediated by the cytoplasmic PABP. We suggest that the 3′-poly(A) tail should be considered a translational enhancer which may stimulate translational initiation in much the same way that transcriptional enhancers are thought to stimulate transcriptional initiation.
Developmentally regulated expression of temporally distinct β-glucosidase isozymes in Dictyostelium discoideum
1987, Developmental Biology
During development of Dictyostelium discoideum, the cellular specific activity of β-glucosidase increases before aggregation, declines to low levels during pseudoplasmodium formation, and increases rapidly during culmination. In addition, two electrophoretically distinct isozymes of β-glucosidase are present at different times of development. Using enzyme-specific monoclonal antibodies, we have shown that changes in the level of enzyme specific activity are closely paralleled by changes in the relative rate of β-glucosidase synthesis in vivo and by corresponding changes in the relative cellular concentration of functional β-glucosidase mRNA. Thus, the developmental synthesis of β-glucosidase is controlled at a pretranslational level. Furthermore, our experiments have demonstrated that both β-glucosidase isozymes consist of a single subunit of identical molecular weight. This result is consistent with the previous finding that both isozymes are encoded by the same gene and suggests that the isozymes differ solely with respect to post-translational modification.
Defining the Intracellular Localization Pathways followed by Lysosomal Enzymes in Dictyostelium discoideum
1987, Methods in Cell Biology
This chapter describes the biochemical and genetic methods used to define both the intracellular pathways followed by lysosomal enzymes and the molecular nature of the “sorting” signals found on the proteins. Using biochemical methods, the lysosomal enzymes α-man and β-glu in Dictyostelium are synthesized as precursor polypeptides that are cotranslationally translocated into the lumen of the endoplasmic reticulum (ER) and N-glycosylated. The precursors move at different rates from the RER to the Golgi complex, where they are sulfated and sorted into two classes. One class, containing the majority of the precursor polypeptides, is directed to lysosomes where they are rapidly cleaved to mature forms of the enzymes. The other class of precursors is rapidly secreted from cells. The different intracellular transport rates for the α-man and β-glu precursors support the existence of a transport-mediating receptor. The genetic approach involved the isolation and characterization of mutants altered in the secretion, modification, processing, and/or localization of lysosomal enzymes. Through biochemical analysis of these mutants, one can identify and characterize the molecular components involved in the processing and transport of this group of enzymes.
Identification and characterization of developmentally regulated mRNP proteins of Dictyostelium discoideum
1986, Developmental Biology
The isolation of poly(A)⁺ polysomal and nonpolysomal RNPs by oligo(dT)-cellulose chromatography has led to the identification of more than 20 polypeptides that bind to the poly(A)⁺ mRNA in growing Dictyostelium cells. Most of these polypeptides were identified in experiments using short-wave UV light (254 nm) to crosslink specifically bound proteins to the RNA. Digestion of the RNPs with ribonucleases A and T₁ prior to their application to oligo(dT)-cellulose permitted the isolation of the 3′ poly(A)-protein complexes. In polysomal RNPs, two major polypeptides, with molecular weights of 31,000 (p31) and 31,500 (p31.5), are bound to poly(A). These proteins can also be purified from cytoplasmic extracts by affinity chromatography on poly(A)-Sepharose. Partial proteolytic digestion of p31 and p31.5 indicates that they are closely related. The UV-crosslinking experiments established that p31 and p31.5 bind to the non-poly(A) segments of mRNA as well. In nonpolysomal RNPs, p31 and a polypeptide with a molecular weight of 29,500 (p29.5) are the major species associated with poly(A). Partial proteolytic digestion of p29.5 indicates that it is closely related to p31 and p31.5. Only small amounts of p29.5 were observed in the polysomal poly(A)-protein complexes. Early in Dictyostelium development, when cellular translation activity is sharply reduced, most of the p29.5, p31 and p31.5 present is selectively degraded. These observations are consistent with a translational role for these proteins.
Initial events involved in the synthesis of the lysosomal enzyme α-mannosidase in Dictyostelium discoideum
1986, Archives of Biochemistry and Biophysics
In Dictyostelium discoideum the lysosomal enzyme α-mannosidase is initially synthesized in vivo as a 140,000 M_r protein which is subsequently processed into two mature acidic glycoproteins of 60,000 and 58,000 M_r. To investigate the initial events involved in the synthesis of this protein, mRNA isolated from growing cells was translated in vitro and the resulting protein products were immunoprecipitated with antibodies prepared against the purified enzyme. Messenger RNA prepared from membrane-bound but not free polysomes directed the synthesis of an immunoprecipitable 120K protein that was identified as the α-mannosidase primary translation product by a variety of criteria. Translation in vitro in the presence of dog pancreas microsomes resulted in the conversion of the 120K primary translation product to a 140K form. This 140K species was not accessible to added trypsin under conditions preserving membrane integrity, suggesting it is sequestered in the lumen of the endoplasmic reticulum following synthesis. Treatment of either the in vitro modified or cellular 140K α-mannosidase precursors with endoglycosidase H resulted in the appearance of proteins 2K larger than the primary translation product. The pulse-labeled cellular precursor and the in vitro processed form have similar isoelectric points as revealed by two-dimensional gel electrophoresis. These results imply that the precursor is N-glycosylated in the endoplasmic reticulum possibly without removal of the signal sequence and that the majority of acidic modifications are added late in the post-translational pathway.

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^☆: This research was funded by Grant PCM77-03970 from the National Science Foundation.

^∗: J.C. was supported by USPHS Medical Genetics Postdoctoral Training Grant GM07131-04.

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Developmental Biology

Abstract

References (9)

Translational control of protein synthesis during early stages of differentiation of the slime mold Dictyostelium discoideum

Cell

The precision of ultraviolet adsorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation

Biochim. Biophys. Acta

Isolation and characterization of the RNA of membrane-bound ribosomes in Dictyostelium discoideum

J. Microbiol

RNA in cytoplasmic and nuclear fractions of cellular slime mold amoebas

J. Cell Biol

Cited by (13)

Ribosomal protein gene expression is cell type specific during development in Dictyostelium discoideum

Tales of poly(A): a review

Developmentally regulated expression of temporally distinct β-glucosidase isozymes in Dictyostelium discoideum

Defining the Intracellular Localization Pathways followed by Lysosomal Enzymes in Dictyostelium discoideum

Identification and characterization of developmentally regulated mRNP proteins of Dictyostelium discoideum

Initial events involved in the synthesis of the lysosomal enzyme α-mannosidase in Dictyostelium discoideum

Brief noteMembrane-bound and free polysomes in Dictyostelium discoideum: I. Isolation and developmental effects on size and distribution☆

Abstract

Cell

Biochim. Biophys. Acta

Isolation and characterization of the RNA of membrane-bound ribosomes in Dictyostelium discoideum

J. Microbiol

RNA in cytoplasmic and nuclear fractions of cellular slime mold amoebas

J. Cell Biol

Brief note
Membrane-bound and free polysomes in Dictyostelium discoideum: I. Isolation and developmental effects on size and distribution☆