Anionic Phospholipids flip by Neo1 regulates Golgi association of Arf-GEF Gea2

ABSTRACT

The Golgi apparatus acts like the cell’s shipping center, ensuring proteins reach their correct destinations. A protein called Neo1 helps maintain the balance of lipids, or fats, within the Golgi membrane by flipping certain types—such as PI4P—to the inner side. This balance, known as lipid asymmetry, is vital for healthy cell function, but how Neo1 influences protein movement at the Golgi is not well understood. This study examined whether Neo1 affects the localization of Gea2, a protein that helps activate other factors responsible for packaging and transporting proteins.  Using yeast cells expressing Gea2 fused to mNeonGreen (Gea2-mNG), we compared its localization in normal cells and in cells with Neo1 mutations. We found that Gea2 remained at the Golgi in normal cells but was greatly reduced in Neo1 mutants, especially those unable to flip PI4P properly. These findings suggest that Neo1’s flipping of PI4P is key for keeping Gea2 at the Golgi. Understanding this process may reveal how disruptions in protein trafficking contribute to human diseases like obesity.

INTRODUCTION.

Overview.

Obesity is not just a prevalent health issue; it is a public health crisis with extreme societal and economic impacts. In fact, efforts to combat obesity-related conditions in the United States account for about $147 billion annually. In the United States alone, obesity affects around 42.4% of adults and 19.3% of youth aged 2-19 years [1]. In addition to direct medical costs of obesity, a number of more indirect costs are part of the overall economic impact of obesity. Tsai et al find that the productivity losses to Shell Oil Company alone due to absenteeism effects of obesity were worth $11.2 million per year [2]. This condition shows how health is deeply connected to society’s economy and structure. These aren’t just personal issues, but ones that affect communities, schools, and even workplaces.

A lot of attention has been given to the environmental and behavioral factors that contribute to the development of these disorders, like diet and genetics. Yet, research suggests that failure in cellular processes may play an important role in the development of these disorders [3]. Protein trafficking is a process where cells move proteins and lipids to the places they need to go inside the cell. It can be thought of as a delivery system. After proteins are made, they don’t just stay in one spot, they must be sent to specific locations like the Golgi apparatus or the cell membrane. If this system is disrupted, proteins and lipids may end up in the wrong place or not be delivered at all, which impacts the cell’s ability to function properly.

Protein Trafficking.

A key part of this system involves the formation of vesicles, which are small compartments (Figure 1). These compartments bud off from one organelle and fuse with another. To direct all the traffic that happens within, cells use coat proteins that assemble on membranes to help shape vesicles and select specific cargo to be transported. Lipids in the membrane also play an important role contributing to the physical curve needed for the vesicle to bud and signaling cues that recruit trafficking machinery. Protein and lipid trafficking are tied to metabolism and fat storage [4]. Defects in trafficking pathways can cause proteins or lipids to mislocalize [5].

The Role of Neo1.

Neo1 is a P4-ATPase (phospholipid flippase) that helps maintain lipid asymmetry at the Golgi [6]. Establishing lipid asymmetry is involved in initiating the process of protein trafficking. By flipping specific phospholipids like PE, PS and PI4P, from the outer to the inner cytosolic leaflet, Neo1 directly influences membrane charge and curvature (Figure 2). It’s known that Gea2 is recruited by phospholipids yet the exact method on how remains unknown.

This makes Neo1 critical for proper protein trafficking. Because Arf1 activation and COPI vesicle formation depend on the proper localization of Arf GEF Gea2 [3]. Examining how Neo1 impacts the position of Gea2 in particular provides insight into how lipid organization protein trafficking at the Golgi [5].

Contribution to Obesity.

In the context of Neo1 flippase and Golgi trafficking, disruption of PI4P lipid flipping can indirectly promote obesity-related mechanisms by disturbing the localization of protein Gea2 [6]. When Neo1 function is impaired, the loss of proper Golgi organization prevents efficient trafficking of enzymes and receptors that control lipid and protein processing. This mislocalization can lead to altered secretion of metabolic regulators, abnormal lipid accumulation in the ER and Golgi, as well as increased cellular stress. Over time, these disruptions in lipid trafficking and signaling mimic the metabolic defects observed in adipocytes during obesity, were misdirected lipid handling and inflammation drive fat storage and insulin resistance.

The Role of Arf1 and GEFs in COPI Vesicle Formation.

COPI vesicle formation depends on the small GTPase Arf1, which must be activated to recruit coat proteins to the Golgi. Activation of Arf1 is controlled by guanine nucleotide exchange factors (GEFs), including Gea1 and Gea2 in yeast. These GEFs catalyze the exchange of GDP for GTP on Arf1, allowing it to bind membranes and start coat assembly. While it is clear that Gea2 is required for COPI vesicle formation, the precise mechanism that recruits Gea2 to the Golgi membrane remains uncertain. Previous studies suggest that PI4P and other phospholipids may regulate Golgi-localized proteins, but whether Gea2 specifically depends on these lipid signals has not been determined [7]. This question remains an open area of investigation.

Study Analysis.

The purpose behind this project is to understand if the negative charge of phospholipids PS, PI4P, and PE impact Gea2’s localization at the Golgi membrane. Although it’s known that Neo1 plays major roles in phospholipid asymmetry, understanding if Neo1-mediated phospholipid flipping regulates the Golgi localization of Gea2 in budding yeast cells is still unclear. The Neo1 mutant neo1-1 (loss of function mutation) serves as a negative control showcasing what happens to Gea2 if no phospholipids are present. The Wildtype Neo1 serves as a positive control showcasing what happens if Gea2 functions properly. We sought to determine the necessity of Neo1 function for Gea2 localization at the Golgi and identify contributions from specific phospholipids on Gea2 membrane localization. We predict that based on their negative charge and signaling functions, the reduction of negative phospholipids (PS, PI4P) at the Golgi using separation of function mutations of Neo1 will result in less Gea2 Golgi-recruitment.

MATERIALS AND METHODS.

Bacterial transformation. 

DH5-α competent E. coli cells (GIBCO)​ ​were used and thawed on ice to keep stable. ​​10 μL of DH5-α E. coli and 100ng of plasmid DNA were added into a microcentrifuge tube. A spectrophotometer​ (DeNovix DS-11+)​ was used to measure DNA concentration. The DH5-α E. coli was then incubated on ice for 10 minutes before heat shocking in a water bath at 45^o C for 45 seconds. Immediately after, the DH5-α was again incubated on ice for 2 minutes. Next, 200 μL of LB (Luria broth) was added to the competent cells. It was then placed in a shaking incubator for 20 minutes.

Following the incubation, 150 μL of the bacteria was then added to an (LB)–Amp plate. The plate was then incubated overnight at 37^o C. Bacterial colonies on the LB-Amp plate were inoculated into 10mL of LB-Amp overnight to create a saturated bacterial culture. The plasmid was isolated using a Qiagen QIAprep Spin Mini-Prep Kit (Hilden, Germany). This plasmid was confirmed to have Gea2-mNG by performing a restriction enzyme digest with enzymes EcoRI-HF and BamHI-HF. After digestion, the product was run on a 1% agarose gel to visualize results.

Yeast Strains and Plasmids.

The plasmid pRS313-GEA2-mNeonGreen (pRS313-GEA2-mNG), encoding Gea2 fused to the fluorescent protein mNeonGreen, was used for yeast transformation. The plasmid contains a HIS3 selection marker for yeast selection and an ampicillin resistance gene for bacterial propagation. 2mL of yeast extract peptone dextrose (YPD) media (2% glucose, 10 g/L yeast extract, 20 g/L peptone) was added into 5 tubes. A wooden dowel was used to touch one yeast colony of each strain to add the yeast to the media to grow overnight. The strains used carry separation-of-function mutations in Neo1 affecting phospholipid flipping, including neo1-1 (loss-of-function), WT Neo1, Neo1(S221L), Neo1(Q193A), and Neo1(Q209G). Existing Neo1 mutant strains were used and engineered to express tagged Gea2.  Its purpose is to create a Fluorescence marker for imaging later. Each strain was then incubated at 30^o C while neo1-1 was incubated at 26^oC due to temperature sensitivity. This was done overnight to grow the yeast strains. The next day, 5 new tubes were used as well as 5 cuvettes in order to test the density (OD₆₀₀) of each strain and do a sub-culture. Sub-culturing was done to ensure the yeast were in the exponential growth phase to promote the insertion of the plasmid later. One blank was created with 1mL of YPD media, while the overnight cultures were diluted 1:10 to measure OD600 by spectrophotometer (DeNovix, Wilmington, DE). Yeast strains were sub-cultured in 3 mL for 4 hours prior to transformation.

Yeast transformation was performed using the LiAc-PEG method as previously described (Gietz & Schiestl, 2007). Yeast strains were transformed with a plasmid encoding fluorescently tagged Gea2-mNeonGreen (Gea2-mNG) and selected on –URA media. Transformants were incubated at 30 °C, while the temperature-sensitive neo1-1 strain was maintained at 26 °C to permit growth of transformed colonies.

Image Analysis.

The cells were prepared for imaging using a micropipette to pipette 10ul of the cells onto a glass slide and cover with a cover slip. Cells were imaged on the DeltaVision Elite Deconvolution Microscope using GFP and mCherry illumination sources to capture the Gea2-mNG and Sec7-mCherry fluorescence, respectively. The images were conservatively deconvoluted on a Deltavision microscope. Images were quantified using ImageJ line scans. The lines were drawn from the outside of the cell (yellow), through two Gea2-mNG puncta (purple), and out the other side of the cell. The line scans resulted in intensity values for the Gea2-mNG signal. Peak-to-cytosolic ratios were calculated by dividing the peak (Gea2-mNG puncta) by the cytosolic signal (signal between the two Gea2-mNG puncta) minus the background (signal outside of the cell) (Figure 3).

RESULTS.

The microscopy images were analyzed alongside the P-values received from line scans. The wild-type strain was used as positive control. It showcased normal localization of a yeast cell under the circumstance that Neo1 flips all the phospholipids correctly. neo1-1 which is the loss of function mutation was used as a negative control. neo1-1 loss of function mutation limits Neo1 from flipping any phospholipids at the Golgi. Fluorescence microscopy revealed that Gea2-mNG strongly localized to the Golgi in wild-type Neo1 cells. In contrast, negatively charged Neo1 mutants showed significantly reduced Gea2 recruitment, with the effect most pronounced when PI4P flipping was impaired (4). Quantitative analysis across three biological replicates (N=60) confirmed these observations, with statistically significant differences (p < 0.0001) between wild-type and mutant conditions. These results demonstrate that negatively charged Neo1-mediated lipid flipping, particularly of PI4P, is critical for proper Golgi localization of Gea2.

DISCUSSION.

These findings identify lipid asymmetry as an important regulator of Gea2 recruitment to Golgi membranes. The reduction in Gea2 localization in Neo1 mutants suggests that negatively charged phospholipids flipped by Neo1 either provide direct binding sites or establish membrane properties required for stable protein trafficking. The particularly strong dependence of PI4P emphasizes its role as a key lipid signal for Arf-GEF recruitment. This is significant because there is more than one PIPs in different locations within the cell. For example, PI3P (phosphatidylinositol 3-phosphate) is found on endosomes, while PI4P is mainly at the Golgi and the plasma membrane. Each type of PIP acts like a “tag” that attracts certain proteins. These proteins have special regions that recognize and stick to only one type of lipid, called effector proteins. If PI4P has multiple different spots inside the cell, it can indicate that singular phospholipids can have multiple yet specific functions. This idea is something that has little to no research on, making it a great gateway for future research.

Since Gea2 is required for activating Arf1 and initiating COPI coat assembly, impaired localization likely disrupts retrograde trafficking between the Golgi and ER. These disruptions interfere with vesicle formation, protein sorting, and cellular homeostasis.  Saccharomyces cerevisiae is widely used as a model organism for studying membrane trafficking because many core components of Golgi organization, phospholipid regulation, and vesicle formation are highly conserved across eukaryotes [8]. Although yeast do not model obesity directly, conserved lipid trafficking and membrane regulatory pathways identified in yeast have provided important insight into cellular processes that influence metabolism and lipid homeostasis in mammalian systems.

Beyond yeast biology, these results point toward broader significance. Flippases and PI4P signaling are conserved across eukaryotes, and dysfunction of Neo1-like proteins could contribute to metabolic disorders [7]. Because lipid transport and signaling regulate energy balance, defects in these processes may underlie condition such as obesity.

Future research should investigate whether Gea2 interacts directly with PI4P or whether Neo1-mediated lipid asymmetry indirectly alters Golgi charge to promote binding. These studies will clarify the precise molecular mechanism of recruitment and help connect trafficking defects to human disease. One way to test this would be to mutate the positively charged amino acid residues of Gea2 that would be available to interact with the negatively charged phospholipids at Golgi membrane. If Gea2 directly binds to PI4P or PS, mutation of those residues would cause the same effect to see with a Neo1 mutant- where there is less Gea2 properly localized to the Golgi.

CONCLUSION.

Neo1-mediated lipid flipping is essential for proper Golgi localization of Gea2, with PI4P having the strongest role. By linking lipid asymmetry to Arf-GEF recruitment, this study uncovered a new layer of regulation in protein trafficking at Golgi. Our findings emphasize how protein trafficking is connected to human diseases, including metabolic disorders such as obesity. It also emphasizes how fundamental membrane processes can shape overall cellular and human health. This research can open the door to new ways to combat issues created by obesity and increase the lively hoods of people overall. This can include certain medications that directly affect protein trafficking or create screenings that will alert physicians of the failure of protein trafficking.

ACKNOWLEDGMENTS.

I would like to thank Briana Markham and members of the Graham Lab for mentoring me and allowing me to spend a summer researching in their lab. I would also like to thank Dr. Nicholas Means, Dr. Nathaniel Freymeyer, Dr. Natalia Wallace, and Dr. Gregory Smith for supporting me.

REFERENCES.

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Posted by buchanle on Thursday, May 14, 2026 in May 2026.

Tags: Neo1, Obesity, Phospholipids, Protein Trafficking, Saccharomyces cerevisiae