Protein synthesis by membrane-bound and free ribosomes of secretory and non-secretory tissues

Treatment of synchronous cultures of Chlamydomonas reinhardtii with chloramphenicol at 4 hr after the beginning of the light phase led to a preferential loss of 70S ribosomes from the 17,000 X gmax supernate. The "lost" 70S ribosomes were found associated with a thylakoid membrane fraction prepared from the 17,000 X gm. pellet. Electron microscopic examinations of this fraction revealed that the 70S ribosomes were bound to the unstacked regions of the thylakoid membranes as polygonal penta-and hexamers. These bound ribosomes were only released by treatment with 500 mM KCI and puromycin, suggesting that both ionic interactions and nascent peptide chains were involved in the ribosome-membrane attachment. Since growth of the thylakoid membranes occurs in the light, it is suggested that bound chloroplast ribosomes function in the synthesis of thylakoid membrane proteins.Eukaryotic ribosomes (80 S) occur in two distinct states within the cytoplasm of animal cells: they are either free or bound to the membranes of the endoplasmic reticulum (1, 2). The relative distribution of the two ribosome classes greatly depends on the cell type. Thus, cells active in the synthesis of secretory proteins contain a large proportion of bound ribosomes (3, 4), whereas cells synthesizing proteins primarily for intracellular use have most of their ribosomes in the free state (5,6). In a limited number of cases, it has been shown that the free and membrane-bound ribosomes are indeed involved in the synthesis of different classes of proteins (3,(7)(8)(9)(10)(11), in agreement with the generalization mentioned above. There is, however, indirect evidence that in mammalian hepatocytes the bound ribosomes synthesize not only secretory proteins but also proteins of the endoplasmic reticulum membrane (12-16).Since chloroplasts contain their own ribosomes (70 S), and since their is evidence that these ribosomes synthesize some of the proteins of the thylakoid membranes (17-20), the question arises as to whether these ribosomes also exist as two distinct classes within the chloroplast. In this paper, we demonstrate that in synchronous cultures of Chlamydomonas reinhardtii a detectable fraction of the chloroplast ribosome population becomes bound to the thylakoid membranes during the light phase of the cell cycle at the time when growth of the thylakoid membranes occurs. The correlation suggests that these bound ribosomes are involved in synthesis of thylakoid membrane proteins. Experiments were performed when the cultures reached a cell density of 1 to 2 X 106 cells per ml. Each culture was divided into two equal portions 4 hr after the beginning of the light phase. To one sample, chloramphenicol was added from a stock solution of 100 mg of chloramphenicol per ml of ethanol to give a final concentration of 100 yg/ml, the control sample receiving an equivalent amount of ethanol (final concentration, 0.1%). Both samples were further incubated in the light for 1 hr, which brought the cultures to the 5th hr of the light phase. At this time, t...

supporting

confidence: 68%

Attachment of Chloroplast Polysomes to Thylakoid Membranes in Chlamydomonas reinhardtii

Chua

Blobel

Siekevitz

et al. 1973

“…The intense OP-puro labeling of vesicles in Paneth cells (Fig. 3A, Bottom) suggests that prematurely terminated, OP-puro-conjugated secretory proteins are translocated into the endoplasmic reticulum (ER) lumen, as described before for puro (28). The same pattern of OP-puro labeling was observed in whole-mount stains of the small intestine (Fig.…”

supporting

confidence: 56%

Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin

Liu

Stoleru

et al. 2011

395

378

Synthesis of many proteins is tightly controlled at the level of translation, and plays an essential role in fundamental processes such as cell growth and proliferation, signaling, differentiation, or death. Methods that allow imaging and identification of nascent proteins are critical for dissecting regulation of translation, both spatially and temporally, particularly in whole organisms. We introduce a simple and robust chemical method to image and affinity-purify nascent proteins in cells and in animals, based on an alkyne analog of puromycin, O-propargyl-puromycin (OP-puro). OP-puro forms covalent conjugates with nascent polypeptide chains, which are rapidly turned over by the proteasome and can be visualized or captured by copper(I)-catalyzed azide-alkyne cycloaddition. Unlike methionine analogs, OP-puro does not require methionine-free conditions and, uniquely, can be used to label and assay nascent proteins in whole organisms. This strategy should have broad applicability for imaging protein synthesis and for identifying proteins synthesized under various physiological and pathological conditions in vivo.

“…In lysates from neuroblastoma cells, the incorporation of [3Hileucine into proteins was linear during 20 min and reached a plateau after 30 min of incubation (Fig. 1B).…”

Section: Methodsmentioning

confidence: 99%

“…The specific activity of native ribosomes (free and membrane-bound) was about twice higher in monolayer than in suspension cells (Table 3). Since it has been reported (14,20) that, in in vitro systems, membrane-bound ribosomes are more active than free ribosomes during short incubation times, the difference in activity of native ribosomes could be due to a change in the relative amounts of free and membranebound ribosomes in the two cell populations. Therefore, neuroblastoma ribosomes were treated with 1% deoxycholate (final concentration), so that all ribosomes would be present as free particles.…”

Section: Cell Sap Activitymentioning

confidence: 99%

Regulation of protein synthesis at the translational level in neuroblastoma cells.

Zucco¹,

Persico²,

Felsani³

et al. 1975

Protein synthesis in neuroblastoma cells has been studied in a cell-free system. The activity of lysates from cells grown in suspension and monolayer has been compared. A higher level of activity has been found in monolayer cells. The activity of some components of the lysate that are involved in protein synthesis has been analyzed. The data suggest that the controlling step of protein synthesis in this system might be the initiation process. The correlation between activation of protein synthesis and neurite outgrowth in monolayer cultures is discussed.Neuroblastoma cells can be grown in two different culture conditions, with morphological aspects resembling different stages of neuronal maturation (1, 2). If neuroblastoma cells are grown in suspension culture, they appear as spherical, immature neuroblasts; in monolayer cultures, when the cells attach to the dish, they become able to extend neurites and, therefore, look like mature neurons. Several biochemical correlates of this "maturation" in vitro have been described, such as increases in ribosomal RNA (3) and cell surface, as well as changes in surface glycoproteins (4, 5) and antigens (6). In addition, changes in functional properties of the cell surface, such as acetylcholine sensitivity, have been reported (7).On the basis of these findings, it seemed of interest to investigate the protein-synthesizing machinery and its control mechanisms during the transition of neuroblastoma cells from suspension to monolayer cultures. We began this investigation by assaying the protein synthesis activity of the two types of neuroblastoma cells under conditions in vivo. Unfortunately no definite results could be obtained because the rate of amino-acid uptake was highly variable in different experiments. For this reason, we decided to approach the problem by using a cell-free protein-synthesizing system. In the present paper we report on studies of protein synthesis in vitro in neuroblastoma cells placed in the two different conditions of growth. Assay of Total Soluble Activity. For measurement of the activity of the soluble factors involved in protein synthesis, the incubation mixture contained 30 ,ug of heterologous, deoxycholate-treated ribosomes from rat brain cortex (10) and increasing amounts of neuroblastoma cell sap. Incubation and further processing were as described for the standard cellfree protein synthesis procedure. The proteins were determined by the method of Lowry et al. (11).Assay of Total Transfer Activity. The activity of the elongation factors I and II was measured as described (12