Golgi Integrity Research Articles

Being or becoming the Golgi: framework or process?

The constancy of biological structures is undeniable, but some structures self-assemble and reach equilibria, whereas others self-organize and attain steady states that are far from equilibrium [ 1 Misteli T. The concept of self-organization in cellular architecture. J. Cell Biol. 2001; 155: 181-185 Crossref PubMed Scopus (380) Google Scholar ]. ‘The processes that occur within a self-organized structure are not underpinned by a rigid architectural framework; rather, they determine its organization.’ Current skirmishes in the Golgi ‘wars’ tilt exactly on this distinction: does a persistent matrix nucleate Golgi assembly or does the organelle self-organize from components as they are exported by the endoplasmic reticulum (ER)? Various agents that block protein export from the ER cause Golgi structure to be lost and Golgi components to relocalize to the ER. Thus, many consider the Golgi a self-organizing, energy-dissipating, dynamic organelle, the components of which are in continual flux between the membranes of the ER and Golgi. Against this tide, Graham Warren's lab has shown that a stable membrane scaffold might nucleate Golgi assembly. They find that, despite loss of Golgi integrity, a membrane compartment retains a specific subset of Golgi ‘matrix’ proteins and persists at the characteristic juxtanuclear Golgi location.

Involvement of ADP-ribosylation Factor 1 in Cholera Toxin-induced Morphological Changes of Chinese Hamster Ovary Cells

ADP-ribosylation factor 1 (ARF1) was originally found as a cofactor in CT-catalyzed ADP-ribosylation of Galpha(s) but is now known to participate in vesicle trafficking. We asked whether ARF1 function in vesicular trafficking is necessary for CT-induced morphological changes in Chinese hamster ovary (CHO) cells, which result from increased intracellular cAMP. Brefeldin A treatment of cells suppressed CT action, confirming a requirement for Golgi integrity. Overexpression of a GFP-ARF1 fusion protein did not affect the morphological changes induced by CT, but changes were reduced in cells overexpressing guanine nucleotide exchange-defective ARF1(T31N) or GTP hydrolysis-deficient ARF1(Q71L) mutants. In cells expressing these mutants, 8-bromo-cAMP induced changes similar to those seen in cells transfected with ARF1 or vector. Inhibition of CT action was specific for mutants of ARF1 and not reproduced by analogous mutants of ARF5 or ARF6. ARF1(Q71L) was mostly colocalized with betaCOP, but ARF5(Q71L) less so. ARF6(Q67L) did not colocalize with betaCOP and was partially associated with the plasma membrane. These data are consistent with the conclusion that ARF1 influenced CT action in cells by its specific function in the vesicular transport pathway used by CT to travel from plasma membrane to Golgi to ER.

RGS4 and RGS2 bind coatomer and inhibit COPI association with Golgi membranes and intracellular transport.

COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit beta'-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant beta'-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of beta'-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on G(ialpha). In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi-plasma membrane or intra-Golgi transport.

Apoptotic Signals at the Golgi

STKE Caspases are proteases that mediate a sequence of events leading to apoptosis or programmed cell death. Mancini et al. show that caspase-2 localizes primarily to the cytoplasmic face of the Golgi complex (which disintegrates during apoptosis) and cleaves golgin-160, a Golgi membrane protein implicated in maintaining Golgi integrity and membrane trafficking. When a caspase-resistant form of golgin-160 was overexpressed in cultured cells, Golgi disintegration was delayed in response to apoptosis-inducing agents. These data support the notion that specific caspases may localize to distinct intracellular sites in the course of transducing apoptotic signals.— LC J. Cell Biol. 149 , 603 (2000).

Apoptotic Signals at the Golgi

Caspases are the proteases that transduce proapoptotic signals in cells. Mancini et al. show that caspase-2 localizes primarily to the cytoplasmic face of the Golgi complex. Casapse-2 cleaved golgin-160, a Golgi membrane protein implicated in maintaining Golgi integrity and membrane trafficking. When a noncleavable mutant form of golgin-160 was overexpressed in cultured cells, Golgi disintegration was delayed in response to apoptosis-inducing agents. The data support the notion that particular caspases may localize to specific intracellular sites to interact with specific substrates and to generate distinct apoptotic signals. Mancini, M., Machamer, C.E., Roy, S., Nicholson, D.W., Thornberry, N.A., Casciola-Rosen, L.A., and Rosen, A. (2000) Caspase-2 is localized at the Golgi complex and cleaves golgin-160 during apoptosis. J. Cell Biol. 149 : 603-612. [Abstract] [Full Text]

Inhibition of secretion by 1,3-Cyclohexanebis(methylamine), a dibasic compound that interferes with coatomer function.

We noted previously that certain aminoglycoside antibiotics inhibit the binding of coatomer to Golgi membranes in vitro. The inhibition is mediated in part by two primary amino groups present at the 1 and 3 positions of the 2-deoxystreptamine moiety of the antibiotics. These two amines appear to mimic the epsilon-amino groups present in the two lysine residues of the KKXX motif that is known to bind coatomer. Here we report the effects of 1, 3-cyclohexanebis(methylamine) (CBM) on secretion in vivo, a compound chosen for study because it contains primary amino groups that resemble those in 2-deoxystreptamine and it should penetrate lipid bilayers more readily than antibiotics. CBM inhibited coatomer binding to Golgi membranes in vitro and in vivo and inhibited secretion by intact cells. Despite depressed binding of coatomer in vivo, the Golgi complex retained its characteristic perinuclear location in the presence of CBM and did not fuse with the endoplasmic reticulum (ER). Transport from the ER to the Golgi was also not blocked by CBM. These data suggest that a full complement of coat protein I (COPI) on membranes is not critical for maintenance of Golgi integrity or for traffic from the ER to the Golgi but is necessary for transport through the Golgi to the plasma membrane.

The role of ankyrin and spectrin in membrane transport and domain formation

Recent discoveries reveal a Golgi-centric spectrin—ankyrin skeleton required for Golgi integrity and anterograde protein trafficking. Identification of specific functional domains in spectrin that mediate its association with motor proteins and the Golgi complex has allowed novel insights into the structure and function of the secretory pathway, and into how this process is controlled by ADP-ribosylation factor and phosphoinositides. Alternative models of Golgi spectrin function that have been recently proposed are reviewed.

A GDP-bound of rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments.

Rab1 is a small GTPase regulating vesicular traffic between early compartments of the secretory pathway. To explore the role of rab1 we have analyzed the function of a mutant (rab1a[S25N]) containing a substitution which perturbs Mg2+ coordination and reduces the affinity for GTP, resulting in a form which is likely to be restricted to the GDP-bound state. The rab1a(S25N) mutant led to a marked reduction in protein export from the ER in vivo and in vitro, indicating that a guanine nucleotide exchange protein (GEP) is critical for the recruitment of rab1 during vesicle budding. The mutant protein required posttranslational isoprenylation for inhibition and behaved as a competitive inhibitor of wild-type rab1 function. Both rab1a and rab1b (92% identity) were able to antagonize the inhibitory activity of the rab1a(S25N) mutant, suggesting that these two isoforms are functionally interchangeable. The rab1 mutant also inhibited transport between Golgi compartments and resulted in an apparent loss of the Golgi apparatus, suggesting that Golgi integrity is coupled to rab1 function in vesicular traffic.