ELECTRON MICROSCOPE STUDY OF MITOCHONDRIAL 60S AND CYTOPLASMIC 80S RIBOSOMES FROM LOCUSTA MIGRATORIA

Abstract

Purified mitochondrial ribosomes (60S) have been isolated from locust flight muscle. Purification could be achieved after lysis of mitochondria in 0.055 M MgCl2. Mitochondrial 60S and cytoplasmic 80S ribosomes were investigated by electron microscopy in tissue sections, in sections of pellets of isolated ribosomes, and by negative staining of ribosomal suspensions. In negatively stained preparations, mitochondrial ribosomes show dimensions of ∼270 x 210 x 215 Å; cytoplasmic ribosomes measure ∼295 x 245 x 255 Å. From these values a volume ratio of mitochondrial to cytoplasmic ribosomes of 1: 1.5 was estimated. Despite their different sedimentation constants, mitochondrial ribosomes after negative staining show a morphology similar to that of cytoplasmic ribosomes. Both types of particles show bipartite profiles which are interpreted as "frontal views" and "lateral views." In contrast to measurements on negatively stained particles, the diameter of mitochondrial ribosomes in tissue sections is ∼130 Å, while the diameter of cytoplasmic ribosomes is ∼ 180–200 Å. These data suggest a volume ratio of mitochondrial to cytoplasmic ribosomes of 1:3. Subunits of mitochondrial ribosomes (40S and 25S) were obtained by incubation under dissociating conditions before fixation in glutaraldehyde. After negative staining, mitochondrial large (40S) subunits show rounded profiles with a shallow groove on a flattened side of the profile. Mitochondrial small subunits (25S) display elongated, triangular profiles.

Characterization of mitochondrial and cytoplasmic ribosomes from Paramecium aurelia.

“…These observations are similar to those made in Tetrahymena (20) and in particular demonstrate certain unique structural features, namely the centrally located dense spot between the two approximately equal subunits and the lobe attached to one of the subunits. It has been reported (17,29) that the mitochondrial ribosomes are smaller than their cytoplasmic counterparts, but our results show that they are larger in Paramecium. It is not clear whether these differences in size are produced by the isolation procedure or whether they exist in vivo.…”

A Mitochondrial Odyssey

“…Surprisingly, they had a very small S-value, 55S to 60S, and they contained RNA of a very small size, almost half of that of the cytosolic ribosomes, which for quite some time made us believe they were degradation products (2). But then in several reports, mitochondrial ribosomal RNAs from mammalian cells were reported to be of a similar small size.…”

Direct imaging electron microscopy (EM) methods in modern structural biology: Overview and comparison with X‐ray crystallography and single‐particle cryo‐EM reconstruction in the studies of large macromolecules

“…Alternatively, the tunnel diameter in the middle part is believed to be approximately 1 nm, which may be too small to be detected by freeze‐etching. However, this does not explain why negative staining of the isolated ribosome or ribosomal large subunit did not reveal the entrance (1.5 nm wide) or exit (2.5 nm wide) of the tunnel (Nonomura et al., ; Kleinow et al., ; Ryabova et al., ; Seiser and Nicchitta, ). The centre hole of the RyR (2 nm wide) was visible by negative staining (Saito et al., ), and that of connexin (1.5 nm wide) was observed by negative staining (Caspar et al., ) and freeze‐etching (Hirokawa and Heuser, ).…”

“…However, despite the feasibility of the models produced based on analytical methods, no reports using direct observation methods have confirmed the presence of a tunnel inside the ribosomal subunit. Although many reports have presented images of negative staining of isolated ribosomes or ribosomal subunits, none have produced images showing a tunnel or hole within the subunits (Nonomura et al., ; Kleinow et al., ; Ryabova et al., ; Seiser and Nicchitta, ). Following acceptance of the ribosomal tunnel model, ribosomal subunits on the native ER membrane were directly visualised for the first time by freeze‐etching (Miyaguchi and Reese, ).…”