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The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export.

ABSTRACT: The release of signal peptideless proteins occurs through nonclassical export pathways and the release of fibroblast growth factor (FGF)1 in response to cellular stress is well documented. Although biochemical evidence suggests that the formation of a multiprotein complex containing S100A13 and Synaptotagmin (Syt)1 is important for the release of FGF1, it is unclear where this intracellular complex is assembled. As a result, we employed real-time analysis using confocal fluorescence microscopy to study the spatio-temporal aspects of this nonclassical export pathway and demonstrate that heat shock stimulates the redistribution of FGF1 from a diffuse cytosolic pattern to a locale near the inner surface of the plasma membrane where it colocalized with S100A13 and Syt1. In addition, coexpression of dominant-negative mutant forms of S100A13 and Syt1, which both repress the release of FGF1, failed to inhibit the stress-induced peripheral redistribution of intracellular FGF1. However, amlexanox, a compound that is known to attenuate actin stress fiber formation and FGF1 release, was able to repress this process. These data suggest that the assembly of the intracellular complex involved in the release of FGF1 occurs near the inner surface of the plasma membrane and is dependent on the F-actin cytoskeleton.

SUBMITTER: Prudovsky I

PROVIDER: S-EPMC2173119 | biostudies-literature | 2002 Jul

REPOSITORIES: biostudies-literature

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The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export.

Prudovsky Igor I Bagala Cinzia C Tarantini Francesca F Mandinova Anna A Soldi Raffaella R Bellum Stephen S Maciag Thomas T

The Journal of cell biology 20020722 2

The release of signal peptideless proteins occurs through nonclassical export pathways and the release of fibroblast growth factor (FGF)1 in response to cellular stress is well documented. Although biochemical evidence suggests that the formation of a multiprotein complex containing S100A13 and Synaptotagmin (Syt)1 is important for the release of FGF1, it is unclear where this intracellular complex is assembled. As a result, we employed real-time analysis using confocal fluorescence microscopy t ...[more]

PMID: 12135982

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Project description:Baculovirus entry into insect midgut cells is dependent on a multiprotein complex of per os infectivity factors (PIFs) on the envelopes of occlusion-derived virions (ODVs). The structure and assembly of the PIF complex are largely unknown. To reveal the complete members of the complex, a combination of blue native polyacrylamide gel electrophoresis, liquid chromatography-tandem mass spectrometry, and Western blotting was conducted on three different baculoviruses. The results showed that the PIF complex has a molecular mass of ∼500 kDa and consists of nine PIFs, including a newly discovered member (PIF9). To decipher the assembly process, each pif gene was knocked out from the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome individually by use of synthetic baculovirus technology, and the impact on PIF complex formation was investigated. Deletion of pif8 resulted in the formation of an ∼400-kDa subcomplex. Deletion of pif0, -4, -6, -7, or -9 resulted in a subcomplex of ∼230 kDa, but deletion of pif1, -2, or -3 abolished formation of any complex. Taken together, our data identified a core complex of ∼230 kDa, consisting of PIF1, -2, and -3. This revised the previous knowledge that the core complex was about 170 kDa and contained PIF1 to -4. Analysis of the PIF complex in cellular fractions suggested that it is assembled in the cytoplasm before being transported to the nucleus and subsequently incorporated into the envelopes of ODVs. Only the full complex, not the subcomplex, is resistant to proteolytic attack, indicating the essentiality of correct complex assembly for oral infection.IMPORTANCE Entry of baculovirus into host insects is mediated by a per os infectivity factor (PIF) complex on the envelopes of occlusion-derived viruses (ODVs). Knowledge of the composition and structure of the PIF complex is fundamental to understanding its mode of action. By using multiple approaches, we determined the complete list of proteins (nine) in the PIF complex. In contrast to previous knowledge in the field, the core complex is revised to ∼230 kDa and consists of PIF1 to -3 but not PIF4. Interestingly, our results suggest that the PIF complex is formed in the cytoplasm prior to its transport to the nucleus and subsequent incorporation into ODVs. Only the full complex is resistant to proteolytic degradation in the insect midgut, implying the critical role of the entire complex. These findings provide the baseline for future studies on the ODV entry mechanism mediated by the multiprotein complex.

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The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export.

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The intracellular translocation of the components of the fibroblast growth factor 1 release complex precedes their assembly prior to export.

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