GORASP2 Human

What is GORASP2 and what is its primary function in human cells?

GORASP2, also known as GRASP55, is a member of the Golgi reassembly stacking protein family that plays a crucial role in maintaining the stacked architecture of the Golgi apparatus. It functions as a "glue" protein that facilitates the stacking of Golgi cisternae and the formation of the Golgi ribbon structure . GORASP2 is a peripheral membrane protein located on the cytoplasmic face of Golgi cisternae that forms homo-dimers which then oligomerize in trans through their conserved N-terminal GRASP domain . This oligomerization process enables GORASP2 to stick adjacent cisternae together into stacks and to link Golgi stacks into a ribbon. The maintenance of this unique Golgi architecture is essential for accurate protein glycosylation and sorting in the secretory pathway .

How is GORASP2 regulated post-translationally?

One key post-translational modification that regulates GORASP2 function is O-GlcNAcylation. Under normal growth conditions, GORASP2 is O-GlcNAcylated, which maintains its localization and function at the Golgi apparatus . Upon glucose starvation, GORASP2 undergoes de-O-GlcNAcylation, triggering its partial relocation from the Golgi to the autophagosome-lysosome interface . This change in modification status acts as a molecular switch that redirects GORASP2's function from Golgi stacking to facilitating autophagosome-lysosome fusion during nutrient stress . This regulatory mechanism illustrates how cells can repurpose existing proteins for alternative functions in response to metabolic challenges, optimizing resource utilization during stress conditions.

What role does GORASP2 play in autophagy during metabolic stress?

During glucose deprivation, GORASP2 adopts an unconventional role in autophagy as a sensor of glucose levels . Upon de-O-GlcNAcylation triggered by glucose starvation, a subpopulation of GORASP2 relocates from the Golgi to the autophagosome-lysosome interface . At this interface, GORASP2 interacts with lipidated LC3 on autophagosomes and LAMP2 on lysosomes, functioning as a membrane tether to facilitate autophagosome-lysosome fusion . Additionally, GORASP2 contributes to multiple steps in the autophagy process:

• It regulates phagophore closure by mediating the association between VPS4A and the ESCRT-III component CHMP2A

• It controls RAB7A activity by modulating its GEF complex, MON1A-CCZ1

• It influences the assembly of SNARE complexes critical for membrane fusion

These multifaceted roles establish GORASP2 as an important coordinator of autophagosome maturation in response to metabolic stress.

What methodologies are most effective for studying GORASP2's role in autophagosome-lysosome fusion?

Several complementary experimental approaches have proven effective for investigating GORASP2's function in autophagosome-lysosome fusion:

• Super-resolution microscopy: Structured illumination microscopy (SIM) has successfully visualized GORASP2 localization on autophagosomal surfaces during glucose starvation

• Protein-protein interaction assays: Co-immunoprecipitation experiments to detect interactions between GORASP2 and autophagy-related proteins (LC3, LAMP2, RAB7A, SNARE components)

• siRNA-mediated knockdown: Depletion of GORASP2 combined with autophagy assays to determine its functional significance

• Fluorescence microscopy: Monitoring autophagosome and lysosome dynamics using fluorescent markers (such as LC3-GFP and LAMP1/2) in cells with normal or depleted GORASP2 levels

• Autophagy flux analysis: Using bafilomycin A1 (BafA1) in combination with glucose starvation to assess autophagosome accumulation versus lysosomal degradation

These methodological approaches provide researchers with a toolkit for dissecting the specific mechanisms through which GORASP2 coordinates autophagosome-lysosome fusion in response to metabolic stress.

How does GORASP2 facilitate phagophore closure during autophagosome formation?

GORASP2 plays a critical role in the final stages of autophagosome formation by regulating phagophore closure . This process involves GORASP2's interaction with components of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery. Specifically, GORASP2 regulates the association between VPS4A and the ESCRT-III component CHMP2A . The ESCRT-III complex is responsible for membrane scission events in various cellular processes, including the final sealing of the phagophore membrane to form a complete autophagosome.

When GORASP2 is depleted, this association is hindered, resulting in defective phagophore closure . This finding indicates that GORASP2 not only functions in the later stages of autophagosome-lysosome fusion but also contributes to the earlier step of autophagosome completion. This role is particularly important because incomplete closure of autophagosomes would prevent their subsequent fusion with lysosomes, thereby disrupting the entire autophagy process. The involvement of GORASP2 in phagophore closure represents an important mechanistic link between autophagosome formation and maturation during cellular stress responses.

What is the relationship between GORASP2 and cellular energy metabolism?

GORASP2 plays a significant role in regulating cellular energy metabolism, particularly in maintaining ATP production. RNA sequencing and KEGG pathway analysis have revealed that GORASP2 knockdown suppresses several key energy metabolism pathways , including:

• Valine, leucine and isoleucine degradation

• Propanoate metabolism

• Fatty acid metabolism

• Citrate cycle (TCA cycle)

• Butanoate metabolism

Quantitative measurements have demonstrated that GORASP2 depletion reduces ATP production levels by approximately 21.9% in human myometrial smooth muscle cells (hMSMCs) . Furthermore, oxygen consumption rate (OCR) analysis has shown that GORASP2 knockdown impairs aerobic respiration, decreasing basal respiration, ATP-production coupled respiration, and maximal respiration capacity in cells .

At the molecular level, GORASP2 regulates the expression of several metabolism-related genes, including ACAT2, ACADM, DLD, and AKT3, which are involved in fatty acid oxidation and mitochondrial function . This connection between a Golgi structural protein and energy metabolism highlights the integrated nature of cellular processes and suggests GORASP2 may serve as a metabolic regulator beyond its canonical role in Golgi organization.

How does GORASP2 affect mitochondrial function and ATP production?

GORASP2 significantly impacts mitochondrial function and ATP production through multiple mechanisms. Experimental evidence demonstrates that GORASP2 knockdown results in:

• Reduced ATP levels: Direct measurement shows a decline of approximately 21.9% in cellular ATP content following GORASP2 depletion

• Impaired aerobic respiration: Oxygen consumption rate (OCR) analysis reveals decreased basal respiration, ATP-production coupled respiration, and maximal respiration elicited by FCCP in GORASP2-depleted cells

• Diminished spare respiratory capacity: GORASP2 knockdown significantly reduces the cell's ability to increase respiration in response to increased energy demand

• Increased mitochondrial ROS: Depletion of GORASP2 leads to elevated mitochondrial reactive oxygen species (ROS) production, as measured using MitoSOX dyes

• Downregulation of key metabolic genes: GORASP2 knockdown reduces expression of critical genes involved in energy metabolism, including those encoding enzymes such as medium-chain acyl-CoA dehydrogenase (ACADM) and dihydrolipoamide dehydrogenase (DLD)

These findings suggest that GORASP2 plays an integral role in maintaining mitochondrial function and energy homeostasis, though the precise molecular mechanism linking this Golgi protein to mitochondrial processes requires further investigation.

What experimental approaches are most suitable for investigating GORASP2's impact on cellular bioenergetics?

Several complementary methodological approaches have proven effective for investigating GORASP2's role in cellular bioenergetics:

• ATP measurements: Quantitative luminescence-based assays to directly measure cellular ATP content following GORASP2 manipulation

• Oxygen Consumption Rate (OCR) analysis: Use of instruments like the Seahorse XF Analyzer to measure key parameters of mitochondrial function, including:

  • Basal respiration

  • ATP-production coupled respiration

  • Maximal respiration (using FCCP)

  • Spare respiratory capacity

• RNA sequencing with pathway analysis: Comprehensive profiling of gene expression changes following GORASP2 knockdown, coupled with KEGG pathway analysis to identify affected metabolic pathways

• RT-qPCR validation: Targeted assessment of expression changes in specific metabolism-related genes (e.g., ACAT2, ACADM, DLD, AKT3)

• Mitochondrial ROS measurements: Use of specific dyes like MitoSOX to assess mitochondrial reactive oxygen species production as an indicator of mitochondrial stress

• siRNA-mediated knockdown: Specific depletion of GORASP2 to assess direct impacts on metabolic parameters

These methodologies provide researchers with a comprehensive toolkit for dissecting the specific mechanisms through which GORASP2 influences cellular energy metabolism at both the functional and molecular levels.

What is the role of GORASP2 in human myometrial contractility during labor?

GORASP2 plays a significant role in regulating myometrial contractility during labor. Western blot analysis has confirmed increased expression of GORASP2 in laboring myometrium compared to non-laboring tissue . Functional studies using siRNA-mediated knockdown of GORASP2 in primary human myometrial smooth muscle cells (hMSMCs) have demonstrated that GORASP2 depletion significantly reduces cell contractility .

Interestingly, this effect on contractility occurs independently of changes in contraction-associated proteins such as OXTR and Connexin 43 . When hMSMCs were subjected to hypoxia (which normally enhances contractions), GORASP2 knockdown partially reversed the enhanced cell contractility, indicating that GORASP2 is necessary for maintaining optimal contractile function during labor-like stress conditions .

The mechanism appears to be primarily related to GORASP2's role in maintaining ATP production rather than through effects on autophagy, as GORASP2 knockdown failed to inhibit hypoxia-induced autophagy in hMSMCs . This connection between GORASP2, energy metabolism, and myometrial contractility suggests that GORASP2 upregulation during labor may be an adaptive response to meet the increased energy demands of uterine contractions.

What are the cellular consequences of GORASP2 depletion in human cells?

GORASP2 depletion results in multiple cellular consequences that extend beyond its canonical role in Golgi organization. Experimental evidence demonstrates that GORASP2 knockdown leads to:

• Reduced cell contractility: In human myometrial smooth muscle cells, GORASP2 depletion significantly decreases cellular contractility under both normoxic and hypoxic conditions

• Impaired energy metabolism: GORASP2 knockdown suppresses multiple energy metabolism pathways and reduces ATP production by approximately 21.9%

• Mitochondrial dysfunction: Cells with depleted GORASP2 show decreased basal respiration, ATP-production coupled respiration, and maximal respiratory capacity

• Increased oxidative stress: GORASP2 knockdown results in elevated intracellular reactive oxygen species (ROS) and specifically increased mitochondrial ROS

• Enhanced apoptosis: Flow cytometry analysis has shown that GORASP2 depletion increases cellular apoptosis rates

• Cell cycle disruption: GORASP2 knockdown causes cell cycle arrest in the S phase

• Defective autophagosome maturation: In certain cell types under glucose starvation, GORASP2 depletion hinders phagophore closure and autophagosome-lysosome fusion

These diverse cellular consequences highlight GORASP2's multifunctional role in maintaining cellular homeostasis across various biological processes.

How do different stress conditions affect GORASP2 localization and function?

GORASP2 exhibits remarkable plasticity in its localization and function in response to different cellular stress conditions:

• Glucose deprivation:

  • Triggers de-O-GlcNAcylation of GORASP2

  • Induces partial relocation from the Golgi to the autophagosome-lysosome interface

  • Repurposes GORASP2 to function as a membrane tether facilitating autophagosome-lysosome fusion

  • Enables GORASP2 to interact with LC3 on autophagosomes and LAMP2 on lysosomes

• Hypoxia:

  • Does not significantly alter GORASP2 expression levels in hMSMCs

  • Does not change GORASP2's effect on contraction-associated proteins (OXTR, Connexin 43)

  • Maintains GORASP2's role in supporting cellular contractility

  • Does not modify GORASP2's relationship with autophagy in hMSMCs

• Rapamycin treatment:

  • Effectively induces autophagy in hMSMCs

  • Does not alter GORASP2's impact on autophagy levels in hMSMCs

These differential responses to various stressors highlight GORASP2's context-dependent functions and suggest that its role may be tailored to specific cellular needs under different stress conditions. The molecular mechanisms that direct GORASP2 to different functions under various stressors remain an important area for further investigation.

How does GORASP2 regulate RAB7A activity and SNARE complex assembly?

GORASP2 plays a sophisticated role in coordinating membrane trafficking by regulating both RAB7A activity and SNARE complex assembly during autophagosome maturation. GORASP2 modulates RAB7A activity by influencing its guanine nucleotide exchange factor (GEF) complex, MON1A-CCZ1 . This regulation impacts RAB7A's interaction with the homotypic fusion and protein sorting (HOPS) complex, which is essential for membrane tethering and fusion events .

In parallel, GORASP2 affects the assembly of two distinct SNARE complexes that are critical for driving the final membrane fusion step between autophagosomes and lysosomes:

• STX17-SNAP29-VAMP8 complex

• YKT6-SNAP29-STX7 complex

The assembly of these SNARE complexes is attenuated in the absence of GORASP2, suggesting that GORASP2 either directly facilitates their formation or creates the optimal microenvironment for their assembly . This dual regulation of RAB7A activity and SNARE complex formation positions GORASP2 as a master coordinator of the membrane fusion machinery required for autophagosome maturation during cellular stress.

What are the current contradictions or gaps in our understanding of GORASP2 function?

Several important contradictions and knowledge gaps exist in our current understanding of GORASP2 function:

• Cell-type Specificity: While GORASP2 regulates autophagy in various cell types during glucose starvation , its knockdown in human myometrial smooth muscle cells (hMSMCs) did not affect autophagy levels, even under hypoxia or rapamycin treatment . This suggests context-dependent functions that vary across cell types.

• Mechanism of Metabolic Regulation: It remains unclear how GORASP2, primarily known as a Golgi structural protein, regulates energy metabolism pathways and mitochondrial function . The mechanistic link between Golgi organization and cellular metabolism requires further elucidation.

• Stress-specific Responses: Different stressors (glucose starvation, hypoxia) appear to trigger distinct GORASP2 responses . The molecular determinants that direct GORASP2 to different functions under various stress conditions remain poorly understood.

• Relationship with GORASP1: While both GORASP1 and GORASP2 function in Golgi stacking, their potential redundancy or unique contributions to non-Golgi functions like autophagy need clarification.

• Regulatory Mechanisms: Beyond O-GlcNAcylation , other potential post-translational modifications and regulatory mechanisms that control GORASP2 function remain to be discovered.

Addressing these knowledge gaps will require integrative approaches combining structural biology, advanced imaging, metabolomics, and in vivo models to fully understand GORASP2's multifaceted functions in cellular homeostasis.