Protein kinase D (PKD) binds to diacylglycerol (DAG) in the trans-Golgi network (TGN) and is activated by trimeric G-protein subunits βγ. This complex then regulates the formation of transport carriers in the TGN that traffic to the plasma membrane in non-polarized cells. Here we report specificity of different PKD isoforms in regulating protein trafficking from the TGN. Kinase-inactive forms of PKD1, PKD2 and PKD3 localize to the TGN in polarized and nonpolarized cells. PKD activity is required only for the transport of proteins containing basolateral sorting information, and seems to be cargo specific.Protein kinase D1 (PKD1) is a serine/threonine kinase that binds to the TGN through its first cysteine-rich domain in a DAG-dependent process 1,2 . Kinase-inactive PKD1 (PKD-KD) induces the formation of TGN tubules containing TGN 46 and furin, proteins that cycle between the TGN and the plasma membrane 3 . Resident enzymes of the TGN, such as sialyltransferase or coat proteins of other transport carriers (COPI or clathrin), are not found in these tubules 3 . Furthermore, PKD1 is specific for the transport of proteins from the TGN to the cell surface in non-polarized cells 3 . PKD2 (ref. 4) and PKD3 (ref. 5) have been identified, but it is unknown whether different PKD isoforms have specificity for different classes of cargo proteins or are functionally redundant.The intracellular distribution and function of PKD2 were examined with glutathione Stransferase (GST)-tagged wild-type (WT) and kinase-dead (KD) proteins expressed in HeLa cells (Fig. 1). The TGN of PKD2-KD transfected cells was tubulated and contained TGN 46 ©2004 Nature Publishing Group 7 Correspondence should be addressed to V.M. (malhotra@biomail.ucsd.edu). 8 These authors contributed equally to this work. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2012 June 12. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript and furin (Fig. 1c, d). Neither resident enzymes of the TGN such as sialyltransferase (Fig. 1d) nor coat proteins (COPI and clathrin; data not shown) were present in these tubules. We examined the effects of PKD2-KD expression on post-Golgi transport of ts-G-GFP (green fluorescent protein-tagged ts045 mutant vesicular stomatitis virus-G protein (VSV-G)), a well-characterized exocytic marker 6 . HeLa cells were co-transfected with cDNAs for ts-G-GFP and GST-PKD2-KD at a ratio of 1:5 (ts-G-GFP to PKD2). After incubation overnight at 4 °C, cells were shifted to 20 °C for 3 h in the presence of cycloheximide to accumulate ts-G-GFP in the TGN, and then transferred to 32 °C to allow the protein to leave the TGN. The percentage of cells (n = 500) with ts-G-GFP on the plasma membrane was quantified at different times after the shift to 32 °C. In cells expressing PKD2-WT, about 50% of the cells expressed ts-G-GFP on the plasma membrane within 10 min of the shift to 32 °C, and by 4...

Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane–bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.

Recent studies have identified histone modifications and suggested a role for epigenetic gene regulation in Trypanosoma brucei. The histone modification H4K10ac and histone variants H2AZ and H2BV localize to probable sites of transcription initiation. Although all T. brucei histones have very evolutionarily divergent N-terminal tails, histone H3 shows conservation with other eukaryotic organisms in 6 of 8 amino acids encompassing lysine 4. Tri-methylation of H3K4 is generally associated with transcription. We therefore generated a specific antibody to T. brucei H3K4me3 and performed chromosome immunoprecipitation and high-throughput sequencing. We show that H3K4me3 is enriched at the start of polycistronic transcription units at divergent strand-switch regions and at other sites of RNA Polymerase II transcription reinitiation. H3K4me3 largely co-localizes with H4K10ac, but with a skew towards the upstream side of the H4K10ac peak, suggesting that it is a component of specific nucleosomes that play a role in Pol II transcription initiation.

The self-assembly of proteins into higher order structures is both central to normal biology and a dominant force in disease. Certain glutamine/asparagine (Q/N)-rich proteins in the budding yeast Saccharomyces cerevisiae assemble into self-replicating amyloid-like protein polymers, or prions, that act as genetic elements in an entirely protein-based system of inheritance. The nuclear pore complex (NPC) contains multiple Q/N-rich proteins whose self-assembly has also been proposed to underlie structural and functional properties of the NPC. Here we show that an essential sequence feature of these proteins—repeating GLFG motifs—strongly promotes their self-assembly into amyloids with characteristics of prions. Furthermore, we demonstrate that Nup100 can form bona fide prions, thus establishing a previously undiscovered ability of yeast GLFG nucleoporins to adopt this conformational state in vivo.