Limits...
Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function.

Chen CY, Ingram MF, Rosal PH, Graham TR - J. Cell Biol. (1999)

Bottom Line: Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment.Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p.These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Delta synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-alpha-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Delta cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

Show MeSH

Related in: MedlinePlus

The drs2Δ mutant accumulates abnormal membrane structures that are similar to Berkeley bodies. Electron micrographs of drs2Δ cells incubated for 2 h at 15°C (A) or kept at 30°C (B) showing numerous double-membrane ring and crescent-shaped structures. Plasma membrane (PM) and vacuoles (V) are labeled. The black arrowhead in A denotes a modestly fenestrated ring structure. The arrowhead in B shows a ring structure with a stacked crescent membrane. For comparison, an electron micrograph of similar structures observed in a chc1Δ cell (C, GPY1103) is shown. Narrower, more fenestrated, and typically incomplete ring structures were also found in wild-type (WT) cells grown at 30°C (D, arrowhead). These wild-type ring structures did not stain as darkly as rings found in the drs2Δ mutant (compare to double arrowhead in A). Bars, 0.2 μm. E, drs2Δ and wild-type cells grown at the indicated temperatures were visualized by transmission electron microscopy. Membrane bound structures from 23–25 randomly selected cells were counted and expressed as an average number of structures per cell section.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2168089&req=5

Figure 5: The drs2Δ mutant accumulates abnormal membrane structures that are similar to Berkeley bodies. Electron micrographs of drs2Δ cells incubated for 2 h at 15°C (A) or kept at 30°C (B) showing numerous double-membrane ring and crescent-shaped structures. Plasma membrane (PM) and vacuoles (V) are labeled. The black arrowhead in A denotes a modestly fenestrated ring structure. The arrowhead in B shows a ring structure with a stacked crescent membrane. For comparison, an electron micrograph of similar structures observed in a chc1Δ cell (C, GPY1103) is shown. Narrower, more fenestrated, and typically incomplete ring structures were also found in wild-type (WT) cells grown at 30°C (D, arrowhead). These wild-type ring structures did not stain as darkly as rings found in the drs2Δ mutant (compare to double arrowhead in A). Bars, 0.2 μm. E, drs2Δ and wild-type cells grown at the indicated temperatures were visualized by transmission electron microscopy. Membrane bound structures from 23–25 randomly selected cells were counted and expressed as an average number of structures per cell section.

Mentions: Morphological studies by electron microscopy revealed that drs2Δ cells accumulated aberrant double-membrane ring and crescent-shaped structures at both 15° and 30°C (Fig. 5A and Fig. B). The double-membrane rings in the drs2Δ cells measured 200–250 nm in diameter (average, 240 nm) and often presented a significant gap between the concentric membranes, which should be equivalent to the luminal space of the cisternae. Representative double-membrane ring structures are marked with white arrowheads in Fig. 5 A. Very similar ring structures also accumulated in the chc1Δ mutant (Fig. 5 C; Payne et al. 1987). These types of structures were never observed in wild-type cells; however, ring structures could be found that were more highly fenestrated and appeared to be breaking down into tubules or vesicles (Fig. 5 D, arrowhead). In fact, similar fenestrated ring structures accumulate dramatically in arf1Δ cells (Gaynor et al. 1998). Some of the double-membrane rings in the drs2Δ cells were also modestly fenestrated (Fig. 5 A, black arrow).


Role for Drs2p, a P-type ATPase and potential aminophospholipid translocase, in yeast late Golgi function.

Chen CY, Ingram MF, Rosal PH, Graham TR - J. Cell Biol. (1999)

The drs2Δ mutant accumulates abnormal membrane structures that are similar to Berkeley bodies. Electron micrographs of drs2Δ cells incubated for 2 h at 15°C (A) or kept at 30°C (B) showing numerous double-membrane ring and crescent-shaped structures. Plasma membrane (PM) and vacuoles (V) are labeled. The black arrowhead in A denotes a modestly fenestrated ring structure. The arrowhead in B shows a ring structure with a stacked crescent membrane. For comparison, an electron micrograph of similar structures observed in a chc1Δ cell (C, GPY1103) is shown. Narrower, more fenestrated, and typically incomplete ring structures were also found in wild-type (WT) cells grown at 30°C (D, arrowhead). These wild-type ring structures did not stain as darkly as rings found in the drs2Δ mutant (compare to double arrowhead in A). Bars, 0.2 μm. E, drs2Δ and wild-type cells grown at the indicated temperatures were visualized by transmission electron microscopy. Membrane bound structures from 23–25 randomly selected cells were counted and expressed as an average number of structures per cell section.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2168089&req=5

Figure 5: The drs2Δ mutant accumulates abnormal membrane structures that are similar to Berkeley bodies. Electron micrographs of drs2Δ cells incubated for 2 h at 15°C (A) or kept at 30°C (B) showing numerous double-membrane ring and crescent-shaped structures. Plasma membrane (PM) and vacuoles (V) are labeled. The black arrowhead in A denotes a modestly fenestrated ring structure. The arrowhead in B shows a ring structure with a stacked crescent membrane. For comparison, an electron micrograph of similar structures observed in a chc1Δ cell (C, GPY1103) is shown. Narrower, more fenestrated, and typically incomplete ring structures were also found in wild-type (WT) cells grown at 30°C (D, arrowhead). These wild-type ring structures did not stain as darkly as rings found in the drs2Δ mutant (compare to double arrowhead in A). Bars, 0.2 μm. E, drs2Δ and wild-type cells grown at the indicated temperatures were visualized by transmission electron microscopy. Membrane bound structures from 23–25 randomly selected cells were counted and expressed as an average number of structures per cell section.
Mentions: Morphological studies by electron microscopy revealed that drs2Δ cells accumulated aberrant double-membrane ring and crescent-shaped structures at both 15° and 30°C (Fig. 5A and Fig. B). The double-membrane rings in the drs2Δ cells measured 200–250 nm in diameter (average, 240 nm) and often presented a significant gap between the concentric membranes, which should be equivalent to the luminal space of the cisternae. Representative double-membrane ring structures are marked with white arrowheads in Fig. 5 A. Very similar ring structures also accumulated in the chc1Δ mutant (Fig. 5 C; Payne et al. 1987). These types of structures were never observed in wild-type cells; however, ring structures could be found that were more highly fenestrated and appeared to be breaking down into tubules or vesicles (Fig. 5 D, arrowhead). In fact, similar fenestrated ring structures accumulate dramatically in arf1Δ cells (Gaynor et al. 1998). Some of the double-membrane rings in the drs2Δ cells were also modestly fenestrated (Fig. 5 A, black arrow).

Bottom Line: Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment.Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p.These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, USA.

ABSTRACT
ADP-ribosylation factor appears to regulate the budding of both COPI and clathrin-coated transport vesicles from Golgi membranes. An arf1Delta synthetic lethal screen identified SWA3/DRS2, which encodes an integral membrane P-type ATPase and potential aminophospholipid translocase (or flippase). The drs2 allele is also synthetically lethal with clathrin heavy chain (chc1) temperature-sensitive alleles, but not with mutations in COPI subunits or other SEC genes tested. Consistent with these genetic analyses, we found that the drs2Delta mutant exhibits late Golgi defects that may result from a loss of clathrin function at this compartment. These include a defect in the Kex2-dependent processing of pro-alpha-factor and the accumulation of abnormal Golgi cisternae. Moreover, we observed a marked reduction in clathrin-coated vesicles that can be isolated from the drs2Delta cells. Subcellular fractionation and immunofluorescence analysis indicate that Drs2p localizes to late Golgi membranes containing Kex2p. These observations indicate a novel role for a P-type ATPase in late Golgi function and suggest a possible link between membrane asymmetry and clathrin function at the Golgi complex.

Show MeSH
Related in: MedlinePlus