Intra-Golgi Protein Transport Depends on a Cholesterol Balance in the Lipid Membrane

Abstract

Transport of proteins between intracellular membrane compartments is mediated by a protein machinery that regulates the budding and fusion processes of individual transport steps. Although the core proteins of both processes are defined at great detail, much less is known about the involvement of lipids. Here we report that changing the cellular balance of cholesterol resulted in changes of the morphology of the Golgi apparatus, accompanied by an inhibition of protein transport. By using a well characterized cell-free intra-Golgi transport assay, these observations were further investigated, and it was found that the transport reaction is sensitive to small changes in the cholesterol content of Golgi membranes. Addition as well as removal of cholesterol (10 ؎ 6%) to Golgi membranes by use of methyl-␤-cyclodextrin specifically inhibited the intra-Golgi transport assay. Transport inhibition occurred at the fusion step. Modulation of the cholesterol content changed the lipid raft partitioning of phosphatidylcholine and heterotrimeric G proteins, but not of other (non) lipid raft proteins and lipids. We suggest that the cholesterol balance in Golgi membranes plays an essential role in intra-Golgi protein transport and needs to be carefully regulated to maintain the structural and functional organization of the Golgi apparatus.

Relationship between phosphatidylinositol 4-phosphate synthesis, membrane organization, and lateral diffusion of PI4KIIα at the trans-Golgi network

“…First, the TGN pool of the enzyme contracted in size by up to 50%, and second, there was a visible vesicularization of the eGFP-PI4KII ␣ -containing membranes. These observations are consistent with the previously described effects of M ␤ CD on TGN membranes ( 24,43,44 ) . In addition, M ␤ CD treatment had dramatic effects on cellular sterol levels as imaged by fi lipin staining ( Fig.…”

“…This differs from others who concluded that differences in protein mobility do not necessarily refl ect confi nement to lipid rafts ( 65 ). In the case of PI4KII ␣ , the differences in protein mobility that we observed can be rationalized by overall M ␤ CDinduced changes to membrane morphology and connectivity ( 24,43,44 ) without the need to invoke the targeting of PI4KII ␣ to lipid rafts. In particular, our photobleaching analysis demonstrated that M ␤ CD-induced changes to membrane structure limit the pool of fl uorophore available to replenish the photobleached spot, and this manifests as a reduced mobile fraction of eGFP-PI4KII ␣ .…”

“…Using this approach, other groups have shown that agonist-sensitive PI4P pools are M ␤ CD-sensitive ( 41 ) and that M ␤ CD delocalizes raft-associated PI4P ( 42 ). In addition to lipid raft disruption, cholesterol removal with M ␤ CD is known to induce a more condensed Golgi morphology ( 43 ), partial vesicularisation of TGN membranes ( 24,44 ), and changes in the lateral mobility of some TGN-selectively photobleached with 60 iterative rounds of illumination with the 488 nm line from a 30 mW Argon laser (Lasos Lasertechnik, Jena, Germany) set to 50% output and 100% transmission. Recovery of the fl uorescence signal in this spot was monitored by acquiring 12-bit images at 0.5 s intervals over a time course of 120 s, with the laser power reduced to 0.…”

Membrane lipids and vesicular traffic

“…Unexpectedly, anterograde transport at the Golgi is exquisitely sensitive to changes in the cholesterol concentration [25,26], possibly because this leads to improper segregation of lipids and proteins [27] or to incorrect disposition of proteins on the cytosolic surface. In yeast, membrane flux through the exocytic pathway is regulated by cytosolic Sec14 [28], a family of cytosolic phosphatidylcholine/phosphatidylinositol transfer proteins that via their dual specificity may act as sensors of lipid composition and adapt lipid metabolism.…”

tGolgin-1 (p230, golgin-245) modulates Shiga-toxin transport to the Golgi and Golgi motility towards the microtubule-organizing centre

“…A consequence of chronic missorting to late endosomes would be eventual depletion from the Golgi of recycling glycosphingolipids and probably concomitant depletion of associated cholesterol (Brown, 1998;Marks and Pagano, 2002;van Meer, 2002). Although glycosphingolipid deficiency per se does not result in Golgi dispersal (Sprong et al, 2001), depletion or enhancement of Golgi cholesterol is known to affect Golgi distribution and intra-Golgi transport (Stüven et al, 2003), as well as the requirement for the kinesin family member KIFC3 in Golgi motility (Xu et al, 2002). Cell-to-cell variation in cholesterol or glycosphingolipid content might explain why only 50-65% of tGolgin-1-depleted cells show Golgi dispersal, and cholesterol depletion might explain the reduced steady-state localization of a glycosylphosphatidylinositol-linked protein to the plasma membrane in cells overproducing a tGolgin-1-derived peptide (Kakinuma et al, 2004).…”