Biosynthesis of intestinal microvillar proteins

Cowell, G M; Danielsen, E. Michael

doi:10.1016/0014-5793(84)81147-3

Cited by 21 publications

(4 citation statements)

References 27 publications

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“…large amounts of keratin mRNAs may be required to preserve cellular architecture and terminal web organization in the presence of a rapid turnover of brush border cytoskeletal proteins [57,58]. Such processes may extend to keratin-related polypeptides and be responsible, at least in part, for the increased complexity of the two-dimensional immunoblots.…”

Section: Discussionmentioning

confidence: 99%

Changes in keratin expression during fetal and postnatal development of intestinal epithelial cells

Calnek

Quaroni

1992

Biochemical Journal

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We have investigated keratin expression in fetal, newborn and adult rat intestines by immunofluorescence staining, immunoblotting of two-dimensional gels and Northern blot analysis of total cellular RNAs. Keratin-type intermediate filaments, composed predominantly of keratin no. 19, were observed already in the undifferentiated stratified epithelium present at 15-16 days of gestation. The marked maturation and differentiation of the epithelium taking place at 18-19 days of gestation was characterized by the appearance of the differentiation-specific keratin no. 21 and by a significant increase in the relative amount of keratin no. 8. The keratin pattern typical of adult villus cells became established at the time of birth, and was marked by a considerable increase in the complexity of the keratin-related polypeptides detected on two-dimensional gels, indicative of extensive post-translational modification of all keratins. Starting at 20 days of gestation there was a major increase in the relative abundance of mRNAs coding for keratin nos. 8, 19 and 21; in contrast, the relative amount of keratin no. 18 mRNA reached a peak shortly after birth and declined to very low levels in adult intestine. These results demonstrated marked changes in keratin expression and post-translational processing taking place at key stages of intestinal development. The appearance of keratin no. 21 in coincidence with the formation of an adult-type brush border and terminal web would be consistent with it having an important role in the organization of the intermediate filament network in the apical cytoplasm of the differentiated intestinal cells.

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Section: Discussionmentioning

confidence: 99%

Changes in keratin expression during fetal and postnatal development of intestinal epithelial cells

Calnek

Quaroni

1992

Biochemical Journal

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“…5), but differed markedly from the faint labelling seen in the lane showing non-specifically aggregated material in the gel. Amongst the major bands immunoprecipitated were the 166000-M, and 140000-M, polypeptides of aminopeptidase N. In addition, many other polypeptides were precipitated; one, of about M , 240000 represents the transient and mature forms of sucrase-isomaltase and iiialLase-glucoamylase, and some, in particular those of M , 105000 (1 10-kDa protein), M , 95000 (villin) and Mr 40000 (actin), are likely to correspond to microvillar cytoskeletal components which are known to become labelled within 10 min [17]. Other bands probably correspond to co-precipitated proteins of various intracellular membranes.…”

Section: Immunoelectrophoretic Isolation Of'nzernbrancs Harbouring MImentioning

confidence: 99%

Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex

Danielsen¹,

Cowell²

1985

Eur J Biochem

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Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolaterdl) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.1 1.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of M , 166000 from the latter fraction was cleaved by trypsin, proteinase K and papain, consistent with an extracellular location of the enzyme at its site of function. In contrast, both the mature form and the transient form of M , 140000 from the Mg2+-precipitated fraction were equally well protected from proteolytic cleavage (in the absence of Triton X-100). This indicates that the basolateral plasma membrane is unlikely to be involved in the post-Golgi transport of newly synthesized aminopeptidase N and suggests instead a direct delivery of the enzyme to the apical plasma membrane. A crude membrane preparation from labelled explants was used in immunoelectrophoretic purification of membranes to determine at what stage during intracellular transport newly synthesized microvillar enzymes are sorted, i. e., accumulated in areas of the membrane from where other proteins are excluded. The transient form of aminopeptidase N was only moderately enriched by immunopurification, using antibodies against different microvillar enzymes, but the mature form was enriched approximately 30-fold from explants, labelled for 30 min. This suggets that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.

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“…Such an assignment has implications not only for its role in brush border structure and function but also for its synthesis, transport, and compartmentalization within the cell. In fact, Cowell and Danielsen [26,27] have recently provided biosynthetic data to suggest that llOK, like villin and actin, probably is not synthesized on the rough ER, as is typical for integral membrane proteins, but rather is synthesized on free ribosomes. This would suggest that the llOK either is a peripheral membrane protein or is inserted into the membrane posttranslationally, as has been demonstrated for the microsomal membrane proteins cytochrome b5 and NADH:cytochrome b5 oxidoreductase [see 26,27 for discussion].…”

Section: Discussionmentioning

confidence: 99%

Reevaluation of the hydrophobic nature of the 110‐kD calmodulin‐, actin‐, and membrane‐binding protein of the intestinal microvillus

Conzelman

Mooseker

1986

J of Cellular Biochemistry

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A complex of calmodulin (CM) and the 110-kD (110K) subunit composes the helical array of cross-bridges linking the microvillus actin filament bundle with the membrane. The hydrophobic properties of the 110K protein, assessed by the detergent phase partitioning assay [Bordier C: J Biol Chem 256:1604, 1981], are highly dependent on the solution conditions used in its isolation. The ATP-dissociable 110K-CM complex [Howe and Mooseker: J Cell Biol 97:974, 1983] exhibits hydrophilic characteristics in this assay. In contrast, the 110K subunit extracted from brush borders by Triton X-100, sodium dodecyl sulfate, and sodium pyrophosphate (detergent-treated 110K) [Glenney JR, Glenney P: Cell 37:743, 1984] behaves as a hydrophobic protein. However, because the soluble hydrophilic 110K-CM can be rendered hydrophobic by treating the complex with the same detergent and salt conditions used in the preparation of detergent-treated 110K, the properties of detergent-treated 110K seem likely to be an effect of the solution conditions on its native conformation, sedimentability, or exposure of binding domains. In addition, the detergent-treated 110K is devoid of calmodulin and no longer exhibits the actin-binding activity characteristic of the ATP-dissociable 110K-CM and of the intact complex in situ. With two partially purified preparations of the 110K subunit exhibiting such dramatically distinct properties, it seems premature to define the nature of the 110K subunit's association with the membrane at this time.

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Biosynthesis of intestinal microvillar proteins

Cited by 21 publications

References 27 publications

Changes in keratin expression during fetal and postnatal development of intestinal epithelial cells

Changes in keratin expression during fetal and postnatal development of intestinal epithelial cells

Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex

Reevaluation of the hydrophobic nature of the 110‐kD calmodulin‐, actin‐, and membrane‐binding protein of the intestinal microvillus

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