Ciliary protein turnover continues in the presence of inhibitors of golgi function: evidence for membrane protein pools and unconventional intracellular membrane dynamics.
The intimate association of the Golgi apparatus with cilia suggests a functional alliance. To explore the relationship between the synthesis and processing of membrane constituents and the turnover or regeneration of cilia, parallel cultures of gastrula-stage sea urchin embryos were pulse-chase labeled with (3)H-leucine in the presence of monensin, brefeldin A, or colchicine. Steady-state labeled cilia were isolated, and the embryos were allowed to regenerate cilia, which were then isolated after the equivalent of two normal regeneration times. Regeneration was absent in colchicine, minimal in monensin, and inhibited about 40% by brefeldin A. Both monensin and brefeldin A effectively inhibited the post-translational processing of prominent phosphatidylinositoylated and palmitoylated membrane proteins and the axoneme-associated transmembrane Spec3 protein, yet most other membrane plus matrix and 9+2 axonemal proteins were labeled to levels indistinguishable from untreated controls. However, total protein analysis of the membrane plus matrix fractions showed a substantial increase in glycoproteins and the calsequestrin-like protein ECaSt/PDI after treatment at steady-state with all three inhibitors and after regeneration in brefeldin A. Other constituents of this compartment, such as membrane-associated tubulin, calmodulin, and a 53-kDa calcium-binding protein, were unchanged. Therefore, inhibition of Golgi function via three different mechanisms left 9+2 protein turnover undiminished but resulted in an accumulation, in the cilium, of already-processed membrane pool constituents and a normally ER-resident protein. A disproportionate elevation of HSP70 suggests that a novel stress response may be involved in inhibiting ciliary regeneration or promoting glycoprotein augmentation.
Stephens
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Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts 02118, USA. rstephen@bu.edu
D000818:Animals; D002923:Cilia / Q000502:physiology*; D004625:Embryo, Nonmammalian / Q000502:physiology; D006023:Glycoproteins; D006056:Golgi Apparatus / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012617:Sea Urchins / Q000196:embryology / Q000502:physiology
D006023:Glycoproteins; D008565:Membrane Proteins