Vivek Malhotra

Protein Kinase D Regulates the Fission of Cell Surface Destined Transport Carriers from the Trans-Golgi Network

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.

Gβγ-Mediated Regulation of Golgi Organization Is through the Direct Activation of Protein Kinase D

Abstract We have shown previously that the βγ subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gβγ is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gβγ directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gβγ-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gβγ with PKD regulates the dynamics of Golgi membranes and protein secretion.

Unconventional secretion of Acb1 is mediated by autophagosomes

Evidence is presented for an unconventional protein secretion pathway that is conserved from yeast to Dictyostelium discoideum in which Acb1 may be sequestered into autophagosomal vesicles, which then fuse (either directly or indirectly) with the plasma membrane (see also the companion paper from Manjithaya et al. in this issue).

Functional genomics reveals genes involved in protein secretion and Golgi organization.

Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.

The Curious Status of the Golgi Apparatus

The available evidence suggests the following working hypotheses. (1) COPII vesicles fuse to form ERGIC clusters, which fuse in turn to form a new Golgi cisterna. (2) Each cisterna progresses through the stack and ultimately fragments into transport vesicles. (3) All resident Golgi proteins are continually recycled via retrograde vesicles. This vesicular transport drives the maturation of Golgi cisternae.Each of these hypotheses raises a new set of questions. First, what nucleates the formation of an ERGIC cluster? Is a cytosolic scaffold involved? How does the cell decide when a given ERGIC cluster has been completed, so that subsequent COPII vesicles will contribute to forming a new cluster? What determines the number of ERGIC clusters that fuse to generate a cisterna? Second, is cisternal progression an active process, or do the cisternae simply diffuse? What signal induces a cisterna to undergo terminal maturation at the TGN? Third, how do COPI vesicles incorporate all of the diverse proteins that reside in the Golgi? Are there multiple classes of COPI vesicles (Orci et al. 1997xBidirectional transport by distinct populations of COPI-coated vesicles. Orci, L, Stamnes, M, Ravazzola, M, Amherdt, M, Perrelet, A, Sollner, T.H, and Rothman, J.E. Cell. 1997; 90: 335–349Abstract | Full Text | Full Text PDF | PubMed | Scopus (296)See all ReferencesOrci et al. 1997)? In addition to COPI vesicles, other vesicles apparently retrieve proteins that cycle between the TGN and later compartments of the endomembrane system (Munro 1998xLocalization of proteins to the Golgi apparatus. Munro, S. Trends Cell Biol. 1998; 8: 11–15Abstract | Full Text PDF | PubMed | Scopus (194)See all ReferencesMunro 1998). How is this TGN recycling pathway integrated with intra-Golgi recycling?These questions, which concern the mechanics of Golgi operation, tend to mask the embarrassing fact that we still do not understand how the structure of the Golgi relates to its function. For example, why are Golgi cisternae organized into stacks in most cell types, but dispersed throughout the cytoplasm in S. cerevisiae? One can only speculate that the sites of cisternal assembly are arranged differently in S. cerevisiae than in other eukaryotes. And why do Golgi cisternae have similar diameters in all cells (Farquhar and Palade 1981xThe Golgi apparatus (complex) - (1954–1981) - from artifact to center stage. Farquhar, M.G and Palade, G.E. J. Cell Biol. 1981; 91: 77s–103sCrossref | PubMedSee all ReferencesFarquhar and Palade 1981)? A possible explanation is that if cisternae were larger, vesicular transport would be too slow to effect maturation. Vesicles bud from the cisternal rims, so the maximal rate of vesicular transport will be proportional to a cisternas diameter, whereas the surface area and volume of a cisterna are roughly proportional to the square of its diameter. Thus, the larger the cisterna, the more slowly its contents will turn over by vesicle-driven maturation.Perhaps the most fundamental mystery concerns the basic organization of the Golgi. Why is this organelle subdivided into cisternae in the first place, and why does the number of cisternae per stack range from about 3 to 30 in various cell types (Mollenhauer and Morre 1991xPerspectives on Golgi apparatus form and function. Mollenhauer, H.H and Morre, D.J. J. Electron Microsc. Tech. 1991; 17: 2–14Crossref | PubMed | Scopus (42)See all ReferencesMollenhauer and Morre 1991)? An interesting theory is that the Golgi stack serves as an iterative recycling device for bringing resident ER proteins back to the ER (Rothman and Wieland 1996xProtein sorting by transport vesicles. Rothman, J.E and Wieland, F.T. Science. 1996; 272: 227–234Crossref | PubMedSee all ReferencesRothman and Wieland 1996). However, resident ER proteins rarely travel beyond the first cisterna or two (Pelham 1995xSorting and retrieval between the endoplasmic reticulum and Golgi apparatus. Pelham, H. Curr. Opin. Cell Biol. 1995; 7: 530–535Crossref | PubMed | Scopus (130)See all ReferencesPelham 1995), so it seems unlikely that cells would need extended Golgi stacks solely to recycle ER proteins. An alternative idea comes from recognizing that the Golgi is a carbohydrate factory, with the cisternal stack being analogous to an assembly line (39xThe Golgi complex (in vitro veritas?) . Mellman, I and Simons, K. Cell. 1992; 68: 829–840Abstract | Full Text PDF | PubMed | Scopus (306)See all References, 28xThe molecular and cell biology of glycosyltransferases. Kleene, R and Berger, E.G. Biochim. Biophys. Acta. 1993; 1154: 283–325Crossref | PubMed | Scopus (173)See all References). Secretory products need to spend enough time on the assembly line for all of the carbohydrate processing steps to be completed. By forcing a cisterna to progress through the entire stack, a cell ensures that the secretory cargo remains in the Golgi long enough to be fully processed (i.e, the stack serves as a “delay timer”). In support of this notion, the number of cisternae in a stack seems to correlate with the complexity of the secretory products being produced by the cell (Becker and Melkonian 1996xThe secretory pathway of protists (spatial and functional organization and evolution) . Becker, B and Melkonian, M. Microbiol. Rev. 1996; 60: 697–721PubMedSee all ReferencesBecker and Melkonian 1996).The new dynamic view of the Golgi may also be applicable to endosomes. Indeed, maturation models have been discussed extensively in the endosome field (24xEndosomes. Helenius, A, Mellman, I, Wall, D, and Hubbard, A. Trends Biochem. Sci. 1983; 8: 245–250Abstract | Full Text PDF | Scopus (248)See all References, 20xMembrane transport in the endocytic pathway. Gruenberg, J and Maxfield, F.R. Curr. Opin. Cell Biol. 1995; 7: 552–563Crossref | PubMed | Scopus (508)See all References). One interpretation of the existing data is that endocytic vesicles fuse homotypically to form early endosomes, which mature into late endosomes and eventually fuse with lysosomes. Hence, many compartments of the endomembrane system seem to be transitory structures that can only be understood in the context of other interacting compartments. So how then do we define the Golgi? Where does it begin and end? Seen in this light, the Golgi acquires a curious status: it is more than just an outgrowth of the ER, yet it is not truly an independent organelle.Is the cisternal maturation model the last word on Golgi organization? Almost certainly not. There is still no consensus about any feature of this model. For example, some investigators propose that COPI vesicles mediate anterograde as well as retrograde transport (Orci et al. 1997xBidirectional transport by distinct populations of COPI-coated vesicles. Orci, L, Stamnes, M, Ravazzola, M, Amherdt, M, Perrelet, A, Sollner, T.H, and Rothman, J.E. Cell. 1997; 90: 335–349Abstract | Full Text | Full Text PDF | PubMed | Scopus (296)See all ReferencesOrci et al. 1997). Even more complicated models emerge from the debate about whether tubular connections between cisternae play a role in transport through the stack (39xThe Golgi complex (in vitro veritas?) . Mellman, I and Simons, K. Cell. 1992; 68: 829–840Abstract | Full Text PDF | PubMed | Scopus (306)See all References, 40xVariations on the intracellular transport theme (maturing cisternae and trafficking tubules) . Mironov, A.A, Weidman, P, and Luini, A. J. Cell Biol. 1997; 138: 481–484Crossref | PubMed | Scopus (117)See all References). In his 1975 monograph, Whaley wrote, “No cellular organelle has been the subject of as many, as long-lasting, or as diverse polemics as the Golgi apparatus.” We are happy to report that this statement is just as true in 1998.‡To whom correspondence should be addressed (e-mail: malhotra@biomail.ucsd.edu).

Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network.

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 non-polarized cells. PKD activity is required only for the transport of proteins containing basolateral sorting information, and seems to be cargo specific.

Protein kinase D: an intracellular traffic regulator on the move

Recent research has identified protein kinase D (PKD, also called PKCmu) as a serine/threonine kinase with potentially important roles in growth factor signaling as well as in stress-induced signaling. Moreover, PKD has emerged as an important regulator of plasma membrane enzymes and receptors, in some cases mediating cross-talk between different signaling systems. The recent discovery of two additional kinases belonging to the PKD family and the plethora of proteins that interact with PKD point to a multifaceted regulation and a multifunctional role for these enzymes, with functions in processes as diverse as cell proliferation, apoptosis, immune cell regulation, tumor cell invasion and regulation of Golgi vesicle fission.

Fragmentation and Dispersal of the Pericentriolar Golgi Complex Is Required for Entry into Mitosis in Mammalian Cells

The pericentriolar Golgi stacks are fragmented and found dispersed in mitotic mammalian cells. Addition of an antibody to the Golgi-associated protein GRASP65 inhibited Golgi fragmentation by mitotic cytosol in permeabilized cells. Microinjecting this antibody or the C-terminal fragment of GRASP65, which contains the antibody binding site, into normal rat kidney cells prevented entry into mitosis. Under these conditions the cells had completed S phase but were not in the prophase stage of mitosis. Fragmentation of the Golgi apparatus by nocodazole or Brefeldin A treatment prior to or post microinjection of the anti-GRASP65 antibody alleviated the block in mitotic entry. Based on our findings, we suggest that the pericentriolar Golgi organization is a sensor for controlling entry into mitosis in mammalian cells.

TANGO1 Facilitates Cargo Loading at Endoplasmic Reticulum Exit Sites

A genome-wide screen revealed previously unidentified components required for transport and Golgi organization (TANGO). We now provide evidence that one of these proteins, TANGO1, is an integral membrane protein localized to endoplasmic reticulum (ER) exit sites, with a luminal SH3 domain and a cytoplasmic proline-rich domain (PRD). Knockdown of TANGO1 inhibits export of bulky collagen VII from the ER. The SH3 domain of TANGO1 binds to collagen VII; the PRD binds to the COPII coat subunits, Sec23/24. In this scenario, PRD binding to Sec23/24 subunits could stall COPII carrier biogenesis to permit the luminal domain of TANGO1 to guide SH3-bound cargo into a growing carrier. All cells except those of hematopoietic origin express TANGO1. We propose that TANGO1 exports other cargoes in cells that do not secrete collagen VII. However, TANGO1 does not enter the budding carrier, which represents a unique mechanism to load cargo into COPII carriers.

The formation of golgi stacks from vesiculated golgi membranes requires two distinct fusion events

We have reconstituted the fusion and assembly of vesiculated Golgi membranes (VGMs) into functionally active stacks of cisternae. A kinetic analysis of this assembly process revealed that highly dispersed VGMs of 60-90 nm diameter first fuse to form larger vesicles of 200-300 nm diameter that are clustered together. These vesicles then fuse to form tubular elements and short cisternae, which finally assemble into stacks of cisternae. We now provide evidence that the sequential stack formation from VGMs reflects two distinct fusion processes: the first event is N-ethyl-maleimide (NEM)-sensitive factor (NSF) dependent, and the second fusion event requires an NSF-like NEM-sensitive ATPase called p97. Interestingly, while the earliest steps in stack formation share some similarities with events catalyzing fusion of transport vesicles to its target membrane, neither GTP gamma S nor Rab-GDI, inhibitors of vesicular protein traffic, inhibit stack formation.