Impact of altered protein structures on the intracellular traffic of a mutated vasopressin precursor from Brattleboro rats

Eur J Biochem. 1989 Jul 1;182(3):621-7. doi: 10.1111/j.1432-1033.1989.tb14871.x.

Abstract

The rat vasopressin precursor, synthesized in the reticulocyte lysate system under the direction of in vitro transcribed mRNA, is processed and correctly delivered to the lumen of added microsomal vesicles. Translation of mRNA for the mutant (Brattleboro) vasopressin precursor which lacks a translational stop codon as a consequence of a frame-shift mutation, gives rise to a mutated protein (B-mutant precursor) with a C-terminal poly(lysine) sequence encoded by the poly(A) tail. Upon addition of microsomal membranes, the mutated precursor has access to the lumen of the vesicles as indicated by removal of the signal peptide; however, the C-terminal part with the poly(lysine) tail remains outside the vesicles as shown by its sensitivity to proteinase K. When a modified RNA, including a stop codon located similarly to that found in the cDNA encoding the normal precursor, is translated in the presence of microsomal membranes, the resulting product (S-mutant precursor) is refractory to proteolysis by exogenously added proteinase K. Analysis of the microsomal membranes indicates, however, that the C-terminus of the S-mutant precursor is still anchored within membranes. For studying the intracellular transport of the mutated precursor Xenopus laevis oocytes were injected with various RNA constructs. To monitor the transport steps from the endoplasmic reticulum to the Golgi compartment an RNA encoding a glycosylation site within the S-mutant precursor sequence was constructed. The resulting GS-mutant precursor is synthesized in the oocyte but not secreted into the incubation medium, completely in contrast to the normal vasopressin precursor which can be detected in the incubation bath 4 h after injection of the respective RNA. The sensitivity of the GS-mutant precursor carbohydrate side chain to endoglycosidase H treatment suggests that the mutated precursor does not reach the Golgi apparatus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosaminidase
  • Animals
  • Biological Transport
  • DNA / metabolism
  • Endoplasmic Reticulum / metabolism
  • Genes
  • Golgi Apparatus / metabolism
  • Intracellular Membranes / metabolism
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
  • Microsomes / metabolism
  • Mutation
  • Oocytes / metabolism
  • Protein Biosynthesis
  • Protein Precursors / genetics*
  • Protein Precursors / physiology
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Brattleboro / genetics
  • Vasopressins / biosynthesis
  • Vasopressins / genetics*
  • Xenopus / genetics

Substances

  • Protein Precursors
  • RNA, Messenger
  • Vasopressins
  • DNA
  • Acetylglucosaminidase
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase