In vitro translation of elastin mRNA and processing of the translated products and the signal sequence of elastin a

Abstract

Chick embryo aorta mRNAs were translated in a gel-filtered reticulocyte lysate. The translated products showed two elastin-related proteins (a and b; relative mass approximately 70 000). The translated elastin a protein was separated essentially free of the b protein by centrifugation and sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis. The a protein was then electroeluted from the SDS-polyacrylamide gel and a partial sequence was determined by automated Edman degradation. The NH2-terminal presequence of the elastin a protein is (formula; see text) The assigned sequence is identical to that reported for the b protein. Translation of chick embryo aorta mRNA in the presence of dog pancreas microsomal membranes segregated the a and b proteins into membrane vesicles and cotranslationally cleaved their respective presequences. The processed a and b elastin proteins were isolated together and NH2-terminal proline positions were determined. These are (formula; see text) These proline positions 4 and 8 are identical to those for NH2-terminal sequence for tropoelastin. This suggests that the signal peptidase removes the 24-residue signal peptide and thus directly generates the tropoelastin sequence, with no NH2-terminal prosequence as the intermediate.

Elastin biosynthesis in chick-embryo arteries. Studies on the intracellular site of synthesis of tropoelastin

“…There have been few biochemical studies of elastin synthesis, translocation and secretion, but the demonstration that the primary translation product of elastin mRNA has a short hydrophobic N-terminal leader sequence not found in the secreted protein (Karr & Foster, 1981;Davidson et al, 1982;Krawetz et al, 1983) is consistent with the signal hypothesis of Blobel and colleagues (for review see Blobel, 1977). Thus it is postulated that the hydrophobic signal is likely to play a role in protein-membrane recognition, resulting in cotranslational vectorial discharge into the lumen of the endoplasmic reticulum (Sabatini et al, 1982).…”

Primary structure and possible origin of the non-glycosylated basic proline-rich protein of human submandibular/sublingual saliva

“…Total RNA was isolated from the glands by the method of Harding et al [21] except that the RNA was purified by repeated resuspension and subsequent precipitation with sodium acetate and ethanol [22]. The polyadenylated RNA was prepared from total RNA by oligo(dT)-cellulose chromatography [23,24].…”

The secretion of tropoelastin by chick-embryo artery cells

“…23(0 the mobilities of the higher-Mr peptides in the limited chymotryptic digest show greatest differences and that most of the low-Mr peptides are common to both polypeptides. Evidence that the proposed extra sequence is not the signal peptide which has been lost from tropoelastin b is indicated by studies Krawetz et al, 1983) demonstrating that tropoelastins a and b have similar N-terminal signal-peptide sequences. This latter observation, taken together with the 'peptide mapping' studies in Fig.…”

“…Previous studies have shown that the elastin mRNA prepared from chick artery cells directs the synthesis of two polypeptides, tropoelastin a and tropoelastin b, when translated in cell-free systems (Foster et al, 1980;Krawetz et al, 1983). When these two closely spaced bands are excised from a polyacrylamide gel and re-electrophoresed individually, they continue to exhibit different electrophoretic mobilities (Fig.…”