When a kinase inactive form of Protein Kinase D (PKD-K618N) was expressed in HeLa cells, it localized to the trans-Golgi network (TGN) and caused extensive tubulation. Cargo that was destined for the plasma membrane was found in PKD-K618N-containing tubes but the tubes did not detach from the TGN. As a result, the transfer of cargo from TGN to the plasma membrane was inhibited. We have also demonstrated the formation and subsequent detachment of cargo-containing tubes from the TGN in cells stably expressing low levels of PKD-K618N. Our results suggest that PKD regulates the fission from the TGN of transport carriers that are en route to the cell surface.

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...

More than 100 mammalian proteins are post-translationally modified by glycosylphosphatidylinositol (GPI) at their C-termini and are anchored to the cell surface membrane via the lipid portion. GPI-anchored proteins (GPI-APs) have various functions, such as hydrolytic enzymes, receptors, adhesion molecules, complement regulatory proteins and other immunologically important proteins. GPI-anchored proteins are mainly associated with membrane microdomains or membrane rafts enriched in sphingolipids and cholesterol. It is thought that association with membrane rafts is important for GPI-APs in signal transduction and other functions. Here, we review recent progress in studies on biosynthesis, remodelling and functions of mammalian GPI-APs.

The inositol moiety of mammalian glycosylphosphatidylinositol (GPI) is acylated at an early step in GPI biosynthesis. The inositol acylation is essential for the generation of mature GPI capable of attachment to proteins. However, the acyl group is usually absent from GPI-anchored proteins (GPI-APs) on the cell surface due to inositol deacylation that occurs in the endoplasmic reticulum (ER) soon after GPI-anchor attachment. Mammalian GPI inositol-deacylase has not been cloned, and the biological significance of the deacylation has been unclear. Here we report a GPI inositol-deacylase-deficient Chinese hamster ovary cell line established by taking advantage of resistance to phosphatidylinositolspecific phospholipase C and the gene responsible, which was termed PGAP1 for Post GPI Attachment to Proteins 1. PGAP1 encoded an ER-associated, 922-amino acid membrane protein bearing a lipase consensus motif. Substitution of a conserved putative catalytic serine with alanine resulted in a complete loss of function, indicating that PGAP1 is the GPI inositol-deacylase. The mutant cells showed a clear delay in the maturation of GPI-APs in the Golgi and accumulation of GPI-APs in the ER. Thus, the GPI inositol deacylation is important for efficient transport of GPI-APs from the ER to the Golgi.Many eukaryotic cell surface proteins with various functions are anchored to the membrane via glycosylphosphatidylinositol (GPI) 1 (1-3). GPI-anchored proteins (GPI-APs) on mammalian cells are usually sensitive to bacterial phosphatidylinositolspecific phospholipase C (PI-PLC), leading to the release of the protein portions. Therefore, PI-PLC is often used as a tool to determine whether proteins are GPI-anchored. In contrast, precursors of the GPI-anchor present in the endoplasmic reticulum (ER) are resistant to PI-PLC due to an acyl chain linked to the 2-position of inositol (4). The inositol ring of GPI is acylated at an early step in GPI biosynthesis by the action of PIG-W protein, an acyltransferase that adds a palmitoyl chain to the inositol of glucosaminyl-phosphatidylinositol, the second intermediate in the pathway (5). The inositol acylation is critical for the attachment of GPI to proteins, because mutant cells defective in PIG-W express only very low levels of GPI-APs (5). It is very likely that the acyl group is required for a later step in the pathway when "bridging" ethanolamine phosphate, which links GPI to the protein, is added to the third mannose to generate mature GPI. Soon after the attachment of GPI to proteins, the inositol is usually deacylated in the ER and becomes sensitive to PI-PLC (6). Human erythrocytes represent an exception, in which the inositol remains acylated, and all the GPI-APs are resistant to PI-PLC (7-9). A possible reason for the lack of deacylation in human erythrocytes is that GPIAPs bearing three acyl chains are more stably associated with the membrane than those bearing two acyl chains, and thus the maintenance of GPI-APs during the long life of erythrocytes is ensured.The enzyme involved in ...

No abstract