Autophagosome formation depends on the small GTPase Rab1 and functional ER exit sites.
ABSTRACT Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double-membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long-standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant-negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)-II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.
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ABSTRACT: For several decades, apoptosis has taken center stage as the principal mechanism of programmed cell death (type I cell death) in mammalian tissues. Autophagic cell death (type II) is characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells. The autophagic process is activated as an adaptive response to a variety of extracellular and intracellular stresses, including nutrient deprivation, hormonal or therapeutic treatment, pathogenic infection, aggregated and misfolded proteins, and damaged organelles. Increasing evidence indicates that autophagy is associated with a number of pathological processes, including cancer. The regulation of autophagy in cancer cells is complex since it can enhance cancer cell survival in response to certain stresses, while it can also act to suppress the initiation of cancer growth. This paper focused on recent advances regarding autophagy in cancer and the techniques currently available to manipulate autophagy.International Journal of Cell Biology 01/2012; 2012:317645.
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ABSTRACT: Coxiella burnetii is a Gram-negative obligate intracellular bacterium. After internalization, this bacterium replicates in a large parasitophorous vacuole that has features of both phagolysosomes and autophagosomal compartments. We have previously demonstrated that early after internalization Coxiella phagosomes interact with both the endocytic and the autophagic pathways. In this report, we present evidence that the Coxiella-replicative vacuoles (CRVs) also interact with the secretory pathway. Rab1b is a small GTPase responsible for the anterograde transport between the endoplasmic reticulum and the Golgi apparatus. We present evidence that Rab1b is recruited to the CRV at later infection times (i.e., after 6 h of infection). Interestingly, knockdown of Rab1b altered vacuole growth, indicating that this protein was required for the proper biogenesis of the CRV. In addition, overexpression of the active GTPase-defective mutant (GFP-Rab1b Q67L) affected the development of the Coxiella-replicative compartment inhibiting bacterial growth. On the other hand, disruption of the secretory pathway by brefeldin A treatment or by overexpression of Sar1 T39N, a defective dominant-negative mutant of Sar1, affected the typical spaciousness of the CRVs. Taken together, our results show for the first time that the Coxiella-replicative niche also intercepts the early secretory pathway.Infection and immunity 10/2010; 79(1):402-13. · 4.21 Impact Factor