PMC

Models of protein transport into membranes within the nucleus.

Genetic constructs, in vitro translation, and membrane copurification. (A) Genetic constructs. Numbers indicate positions of amino acids based upon ODV-E66. (B) In vitro translation and membrane copurification. P, microsomal membrane pellet fraction; S, supernatant fraction.

Comparison of N-terminal domains of ODV-E66, ODV-E25, and fusion constructs. Blocked and shaded amino acids indicate hydrophobic domain; underlined (dashed) amino acids were directly N-terminal sequenced; underlined (solid) amino acids indicate N-, S-rich region, boxed amino acids indicate amino acids added by cloning strategy; asterisk indicates charged amino acids. OpMNPV, Orgyia pseudotsugata nuclear polyhedrosis virus; BmMNPV, Bombyx mori nuclear polyhedrosis virus.

ImmunoGold localization of ODV-E25, 23β-gal, 23GFP, 24GFP, and ODV-E66. All data are presented from Sf9 infected cells at 48 hr postinfection and secondary antibody linked to gold. (A) 23β-gal recombinant virus (anti-β-gal); (B) AcMNPV (E2 strain; anti-ODV-E25 1:1000). (C) 23GFP recombinant virus (anti-GFP). (D) 24GFP recombinant virus (anti-GFP). (E) pVL-E66 recombinant virus (anti-ODV-E66, ref. ). n, nucleus; C, cytoplasm; m, microvesicles; ➯, labeling of ODV envelope; ➤, labeling of the nuclear envelope; →, labeling of cytoplasmic membranes; ↔, condensation of cytoplasmic membranes to the periphery of the nucleus. (Bar = 1 μm.)

Fluorescence and ImmunoGold (Janssen) localization of pVL1393–23GFP infected cells. (A and B) 23GFP autofluorescence in pVL1393-23GFP-infected Sf9 cells. (a and b) GFP autofluorescence and phase contract double exposure. (A and a) Forty-eight hours postinfection. (B and b) Seventy-two hours postinfection. Exposure time for cells at different time points postinfection was constant and set for exposure of the 72-hr postinfection. (C) ImmunoGold labeling (48 hr postinfection) using antiserum to GFP (1:1500; CLONTECH) and secondary gold conjugate. Fixation and ImmunoGold labeling of infected were done as described in Hong et al. (). (), Intranuclear microvesicles, (↔), cytoplasmic membranes condensed near the nuclear envelope. Protein was detected as in C. C, cytoplasm; n, nucleus.