First responder to starvation: microreticulophagy clears aberrant membrane proteins in quick bites

ABSTRACT

Cells can use two different pathways for recycling their non-essential components in the lysosome during nutritional stress: macroautophagy and microautophagy. While the well-established macroautophagy pathway requires de novo formation of the double-membrane autophagosome, microautophagy involves direct engulfment of cargo by the lysosomal membrane. Recently, using a yeast model, we identified a novel microreticulophagy pathway induced by nutritional stress that selectively clears aberrant membrane proteins that accumulate during normal growth. This effective clearance occurs rapidly and precedes the degradation of normal ER- or mitochondrial-membrane proteins by macroautophagy. We showed that the nutritional-stress induced selective microreticulophagy pathway requires the ubiquitin-ligase Rsp5, its adaptor Ssh4, and the ESCRT complex. Moreover, live-cell fluorescence microscopy with high temporal and special resolution demonstrated that individual microautophagy events occur within seconds. Thus, cells use the effective microreticulophagy pathway to dispose of misfolded or excess membrane proteins as a first response to starvation. If the stress persists, the more costly macroautophagy pathway is activated for degrading normal cellular components. These findings point to an intricate interplay between microautophagy and macroautophagy during nutritional stress, which optimizes stress responses and could have significant implications for understanding how cells maintain homeostasis or progress to disease states.

Abbreviation: ER, endoplasmic reticulum; ERAD, ER-associated degradation; QC, quality control; reticulophagy, selective autophagy of the ER

Prelude

Constitutive er-quality control during normal cell growth

Two endoplasmic-reticulum quality control (ER-QC) processes are used by cells during normal growth to degrade aberrant membrane proteins: ER-associated degradation (ERAD) and macroreticulophagy. Misfolded proteins are degraded by the ubiquitin-proteasome system in ERAD, whereas macroreticulophagy delivers excess membrane proteins through autophagosomes for degradation in the lysosome. In cells defective in ERAD or macroreticulophagy, misfolded or excess membrane proteins, respectively, accumulate during normal cell growth. We showed that none of the three known stress-signaling pathways – nutritional, oxidative, or heat – is required for these processes to occur, thus establishing that ER-QC during normal growth is constitutive. Moreover, stress-signaling pathways are not induced during normal growth even when aberrant cargo accumulates in ER-QC-defective cells.

The novel microreticulophagy pathway

Nutritional stress induces selective clearance of aberrant membrane proteins

Cells exposed to stress respond by induction of a stress-specific adaptive response. Notably, while oxidative and heat stresses result in accumulation of aberrant proteins, nutritional stress does not have such an effect. Instead, two different autophagy pathways are induced through the TORC1-signaling pathway: macroautophagy and the less-studied microautophagy. Using a yeast model, we showed that exposing cells to nutritional, but not oxidative or heat, stress, can induce effective clearance of aberrant membrane proteins that accumulated during normal growth [1]. This clearance occurs in the lysosome even in mutants defective in macroautophagy, implicating microautophagy as the clearance pathway. Importantly, this pathway is selective to aberrant membrane proteins, because degradation of normal membrane proteins occurs much later through macroautophagy.

Swift and effective clearance during nutritional stress

Most of the selective clearance of aberrant membrane proteins through microautophagy happens within the first two hours after the beginning of the stress. During this time, >50% of misfolded and excess proteins are degraded; hence our dynamic studies focused on the first 30 minutes after exposing the cells to nutritional stress. In contrast, slower-acting macroautophagy degrades only a small fraction of normal membrane proteins at 4 hours. We propose that the first reaction of cells to nutritional stress is to clear non-essential components before degrading normal proteins. In addition, this rapid-response microautophagy pathway does not require resource-intensive synthesis of multiple new components, whereas the macroautophagy pathway relies on the expression of Atg proteins and de novo formation of a double-membrane phagophore that matures into an autophagosome. This explains why cells use microautophagy as a first response, preferably with components already on hand.

Mechanisms of nutritional stress-induced microreticulophagy

The pathway is under the control of TORC1 signaling. Without rapamycin, which inhibits TORC1, the ER cargo does not get close to the lysosomal membrane for its subsequent engulfment. The players we identified as required for the pathway seem to be logical choices and are available in cells during normal growth. First, the ESCRT complex is the only cellular machinery that can seal membrane invaginations. Indeed, without ESCRT, ER loaded with aberrant membrane cargo accumulates in close vicinity to the vacuolar membrane, separating the pathway into two sequential steps dependent on rapamycin and ESCRT (Figure 1). Second, the UB-ligase Rsp5 is known to function with ESCRT and its adaptor Ssh4 allows Rsp5 to recognize other membrane proteins. We found that they are both required for getting the cargo to the vacuole.

Figure 1.

TORC1-induced degradation of membrane proteins during nutritional stress. Upon nutritional stress, inhibition of TORC1 signaling induces two types of pathways that degrade membrane proteins. Left: we identified a novel microreticulophagy pathway induced immediately after exposure to the stress that selectively clears aberrant membrane proteins (red) that accumulated in cells during normal growth. In this pathway, the ER and lysosome get into close proximity, the cargo, an ER fragment with aberrant membrane proteins, is engulfed by an invagination in the lysosomal membrane with the help of the Rsp5 ubiquitin ligase and its Ssh4 adaptor, and the invagination is sealed by ESCRT. Right: the macroreticulophagy pathway is activated later and requires expression of atg proteins and formation of the double-membrane autophagosome (AP). The phagophore membrane engulfs normal membrane proteins (blue) and matures into an AP; the sealed AP fuses with the lysosomal membrane.

Capturing rapid individual microautophagy events

In prior studies, direct engulfment of cargo by the lysosomal membrane in microautophagy, before it enters the lysosome, has been inferred from static images of fixed cells that show the presumed microautophagy stages, usually using electron microscopy for adequate spatial resolution. The use of live cell fluorescence microscopy to observe the stages in real time has been hampered by inadequate temporal or spatial resolution. Therefore, we developed a live-cell microscopy approach with sufficient spatial and temporal resolutions to document the dynamics of microautophagy stages and surprisingly found that individual microautophagy events occur within seconds.

Future

Open mechanistic questions

The mediators of the novel pathway we identified could be recruited from other cellular processes and are available during normal cell growth, thus allowing for such a swift response. Our findings have raised additional fundamental questions about the mechanisms by which aberrant membrane proteins are recognized: Are the aberrant membrane proteins organized in ER membrane domains? What transports the cargo ER to the lysosomal membrane? How are the mediators rapidly recruited during nutritional stress and is this recruitment unique to microreticulophagy?

Extension to human cells and disease

Both ESCRT and Rsp5 have human homologs, and it is likely that this pathway is conserved in human cells to clear disease-associated aberrant membrane proteins. A presence of this pathway in human cells would raise important new questions, such as whether microreticulophagy could be activated to clear toxic or disease-related aberrant proteins by inducing a “clean nutritional stress”, which, unlike other stresses, does not add to the proteotoxic burden cells already have. Conversely, defective microreticulophagy in human cells could prevent the restoration of homeostasis and instead promote disease progression.

Funding Statement

This research was supported by grants [GM-45444 and GM-141479] from NIH to N. Segev.

Disclosure statement

No potential conflict of interest was reported by the author(s).