Autophagy is an evolutionary conserved process by which cells break down components of itself to maintain homeostasis. This process is especially prominent in cells under stress and nutrient deprived conditions. Autophagy involves the formation of membrane bound vesicles called autophagosomes, that engulf cargo and transports it to the lysosomes where breakdown happens. Dysfunctional autophagy, and its dysregulation, has been linked to severe human disorders such as cancer and neurodegeneration thus highlighting the importance of understanding how the process is regulated. One particular gap of knowledge surrounds the lipids that regulate autophagosome genesis. Previous studies have indicated that autophagosomes are cholesterol poor membranes1, suggesting that depletion of cholesterol in autophagosome precursor membranes possibly could generate flexible, highly curved membranes, known to be required for autophagosome biogenesis2. However, a few studies have also found that high cholesterol levels can promote autophagy. Most such studies have involved long term manipulation of cholesterol levels and therefore cannot exclude indirect effects of cholesterol levels.
We hypothesized that local cholesterol depletion from autophagy precursor membranes promotes generation of curved membranes during de novo synthesis of autophagosomal membranes. To study this we first chose to manipulate cholesterol levels in shorter timeframes by treating cells with methyl-β cyclodextrin (MBCD), a commonly used drug to deplete cholesterol. Interestingly, we found that short term cholesterol depletion (15-30 min) caused a rapid induction of autophagy and a corresponding increase in autophagy initiation events. Moreover, short term cholesterol depletion further increased starvation-induced autophagic flux. By using fluorescent markers to examine the curvature of the membranes in real time, it was observed that cholesterol depletion induces the recruitment of curved membrane markers and other crucial proteins involved in the biogenesis of autophagosomes. As autophagosomes are predicted to form from membranes closely associated with the endoplasmic reticulum (ER), we speculated that the membrane curvature needed for recruitment of early autophagy machinery components is achieved by regulating cholesterol levels in the ER.
In order to identify cholesterol transport proteins that might regulate cholesterol levels in autophagosome precursor membranes, we decided to target the GRAMD protein family, which consist of ER anchored transmembrane proteins3, known to mediate plasma membrane to ER cholesterol transport4. By using siRNA mediated knockdown, various GRAMD proteins were depleted in cells expressing an autophagy reporter. Intriguingly, the depletion of GRAMD1C was found to promote starvation-induced autophagic flux, suggesting that GRAMD1C functions as a negative regulator of autophagosome formation. Further experiments demonstrated that both the GRAM domain and the cholesterol transport VASt domain of GRAMD1C are required for its autophagy regulating function. We were able to demonstrate that the GRAM domain of GRAMD1C interacts with mitochondria, in line with the existence of several mitochondrial proteins in the GRAMD1C interactome. The yeast ortholog of GRAMD1C, Lam6, interacts with mitochondria via binding to the outer mitochondrial membrane protein Tom705, and we could confirm that the GRAM domain of GRAMD1C also interacts with TOMM70A.
Having found that the ER localized cholesterol transport protein GRAMD1C interacts with mitochondria, we next asked if GRAMD1C regulates ER-mitochondrial cholesterol transport. Indeed, cells lacking GRAMD1C were found to have increased mitochondrial cholesterol levels, implying that GRAMD1C facilitates transport of cholesterol between the mitochondria and the ER. In line with this, expression of genes involved in cholesterol synthesis was increased in GRAMD1C depleted cells, indicating a corresponding decrease in ER cholesterol levels. Thus, we propose a model where GRAMD1C contributes to the suppression of autophagosome biogenesis by modulating cholesterol levels at ER membranes that are associated with autophagosome initiation sites. As the GRAM domain of GRAMD1C interacts with mitochondria and the plasma membrane, it is likely that GRAMD1C-medaited cholesterol transport from these cellular structures to the ER regulates autophagosome biogenesis.
To investigate whether the increased mitochondrial cholesterol levels seen in GRAMD1C depleted cells affects the mitochondrial bioenergetics, we measured ATP-production linked respiration and the maximal respiratory capacity. Indeed, both were significantly increased in cells lacking GRAMD1C, suggesting that GRAMD1C is a negative regulator of mitochondrial cholesterol abundance and mitochondrial bioenergetics.
In order to investigate whether changes in GRAMD1C levels may be linked to any pathophysiological conditions, we turned to analysis of gene expression data from the TCGA Kidney Renal Clear Cell Carcinoma (KIRC) cohort. Interestingly, GRAMD1C transcript levels is positively correlated with overall survival of patients having Clear Cell Renal Carcinoma (ccRCC), a subtype of kidney cancer characterized by altered mitochondrial metabolism and aberrant lipid and cholesterol accumulation6,7. Further downstream analysis revealed association of expression of several GRAMD family members with patient survival. Additionally, we found that GRAMD1C is co-expressed with several mitochondrial genes in ccRCC samples, suggesting that members of the GRAMD family may contribute to the regulation of overall survival of ccRCC patients through modulation of mitochondrial metabolism.
Taken together, our study underlines the importance of intracellular cholesterol transport in regulation of autophagosome biogenesis and mitochondrial bioenergetics and provides novel insight into how the cholesterol transport protein GRAMD1C functions as a negative regulator of both processes. Moreover, we highlight the importance of GRAMD family proteins in ccRCC development and patient outcome.
Figure 1. GRAMD1C localizes to the ER and interacts with mitochondria via its GRAM domain to facilitate cholesterol transport from mitochondria to the ER. By maintaining cholesterol levels at ER membranes, GRAMD1C likely contributes to the suppression of autophagosome biogenesis. Upon loss of GRAMD1C expression, depletion of cholesterol results in modulation of ER membrane curvature followed by recruitment of early autophagic markers. Concomitant increased cholesterol levels in the mitochondria affects mitochondrial respiration, potentially contributing to the decreased survival of ccRCC patients with low GRAMD1C levels. Figure created with Biorender.
1 Punnonen, E.-L., Pihakaski, K., Mattila, K., Lounatmaa, K. & Hirsimäki, P. Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver. Cell and Tissue Research 258, 269-276 (1989). https://doi.org:10.1007/BF00239447
2 Nath, S. et al. Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3. Nat Cell Biol 16, 415-424 (2014). https://doi.org:10.1038/ncb2940
3 Sandhu, J. et al. Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells. Cell 175, 514-529.e520 (2018). https://doi.org:10.1016/j.cell.2018.08.033
4 Naito, T. et al. Movement of accessible plasma membrane cholesterol by the GRAMD1 lipid transfer protein complex. eLife 8, e51401 (2019). https://doi.org:10.7554/eLife.51401
5 Elbaz-Alon, Y. et al. Lam6 Regulates the Extent of Contacts between Organelles. Cell Reports 12, 7-14 (2015). https://doi.org:https://doi.org/10.1016/j.celrep.2015.06.022
6 Hao, H. et al. Reduced GRAMD1C expression correlates to poor prognosis and immune infiltrates in kidney renal clear cell carcinoma. PeerJ 7, e8205 (2019). https://doi.org:10.7717/peerj.8205
7 Drabkin, H. A. & Gemmill, R. M. Cholesterol and the development of clear-cell renal carcinoma. Curr Opin Pharmacol 12, 742-750 (2012). https://doi.org:10.1016/j.coph.2012.08.002