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

E M Danielsen; G M Cowell

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

E M Danielsen, G M Cowell

Institut for Cellulær og Molekylær Medicin

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

24 Citationer (Scopus)

Abstract

Udgivelsesdato: 1985-Oct-15

Originalsprog	Engelsk
Tidsskrift	European Journal of Biochemistry
Vol/bind	152
Udgave nummer	2
Sider (fra-til)	493-9
Antal sider	6
ISSN	0014-2956
Status	Udgivet - 1985

Bibliografisk note

Keywords: Aminopeptidases; Animals; Antigens, CD13; Biological Transport; Cell Membrane; Endopeptidases; Golgi Apparatus; Immunoelectrophoresis; Intestinal Mucosa; Membrane Proteins; Microvilli; Organ Culture Techniques; Swine

Citationsformater

@article{6005a200e7bd11ddbf70000ea68e967b,

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

abstract = "Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolateral) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.11.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of Mr 166 000 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 Mr 140 000 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 suggests that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.",

author = "Danielsen, {E M} and Cowell, {G M}",

note = "Keywords: Aminopeptidases; Animals; Antigens, CD13; Biological Transport; Cell Membrane; Endopeptidases; Golgi Apparatus; Immunoelectrophoresis; Intestinal Mucosa; Membrane Proteins; Microvilli; Organ Culture Techniques; Swine",

year = "1985",

language = "English",

volume = "152",

pages = "493--9",

journal = "FEBS Journal",

issn = "1742-464X",

publisher = "Springer Verlag",

number = "2",

}

TY - JOUR

T1 - Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex

AU - Danielsen, E M

AU - Cowell, G M

N1 - Keywords: Aminopeptidases; Animals; Antigens, CD13; Biological Transport; Cell Membrane; Endopeptidases; Golgi Apparatus; Immunoelectrophoresis; Intestinal Mucosa; Membrane Proteins; Microvilli; Organ Culture Techniques; Swine

PY - 1985

Y1 - 1985

N2 - Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolateral) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.11.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of Mr 166 000 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 Mr 140 000 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 suggests that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.

AB - Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolateral) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.11.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of Mr 166 000 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 Mr 140 000 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 suggests that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.

M3 - Journal article

C2 - 2865138

VL - 152

SP - 493

EP - 499

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 2

ER -