Growth and form of secretory granules involves stepwise assembly but not differential sorting of a family of secretory proteins in Paramecium.
Paramecium trichocysts are voluminous secretory vesicles consisting of a spindle-shaped body surmounted by a tip that serves to anchor them at exocytotic sites in the plasma membrane. This constrained shape is conferred by the proteins stored in the vesicles, which form an insoluble three-dimensional crystalline array. The constituent polypeptides (Trichocyst Matrix Proteins, TMPs), which assemble during trichocyst biogenesis, are produced by proteolytic processing of soluble proproteins encoded by a large multigene family. In order to investigate the functional significance of the TMP multigene family, which assures the synthesis of a mixture of related polypeptides, we have designed synthetic genes for heterologous expression of three different mature polypeptides, which were used to obtain sequence-specific rabbit antisera. We used these antisera to carry out immunolocalization experiments with wild-type trichocysts at different stages of development and found that the trichocyst matrix consists of two concentric layers containing different TMPs, and that the assembly of each layer corresponds to a distinct phase of trichocyst growth. Examination of mutant trichocysts created by targeted gene silencing of different TMP genes showed that the layer containing the products of the silenced genes is specifically affected, as are all subsequently assembled parts of the structure, consistent with an ordered assembly pathway. This stepwise assembly is not controlled by differential sorting of the TMPs, as single and double label experiments provided evidence that the different TMPs are delivered together to post-Golgi vesicles and developing trichocysts. We present a model for trichocyst biogenesis in which TMP assembly is controlled by protein processing.
Vayssié
L
L
Centre de Génétique Moléculaire, CNRS, France.
Garreau de Loubresse
N
N
Sperling
L
L
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D017931:DNA Primers; D005089:Exocytosis; D020868:Gene Silencing; D005813:Genes, Synthetic; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D005810:Multigene Family; D010247:Paramecium / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D010641:Phenotype; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D022142:Secretory Vesicles / Q000648:ultrastructure*; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D017931:DNA Primers; D015800:Protozoan Proteins