Abstract
HRES-1/Rab4 is a small GTPase that regulates endocytic recycling. It has been colocalized to mitochondria and the mechanistic target of rapamycin (mTOR), a suppressor of autophagy. Since the autophagosomal membrane component microtubule-associated protein light chain 3 (LC3) is derived from mitochondria, we investigated the impact of HRES-1/Rab4 on the formation of LC3+ autophagosomes, their colocalization with HRES-1/Rab4 and mitochondria, and the retention of mitochondria during autophagy induced by starvation and rapamycin. HRES-1/Rab4 exhibited minimal baseline colocalization with LC3, which was enhanced 22-fold upon starvation or 6-fold upon rapamycin treatment. Colocalization of HRES-1/Rab4 with mitochondria was increased >2-fold by starvation or rapamycin. HRES-1/Rab4 overexpression promoted the colocalization of mitochondria with LC3 upon starvation or rapamycin treatment. A dominant-negative mutant, HRES-1/Rab4S27N had reduced colocalization with LC3 and mitochondria upon starvation but not rapamycin treatment. A constitutively active mutant, HRES-1/Rab4Q72L showed diminished colocalization with LC3 but promoted the partitioning of mitochondria with LC3 upon starvation or rapamycin treatment. Phosphorylation-resistant mutant HRES-1/Rab4S204Q showed diminished colocalization with LC3 but increased partitioning to mitochondria. A newly discovered C-terminally truncated native isoform, HRES-1/Rab41–121, showed enhanced localization to LC3 and mitochondria without starvation or rapamycin treatment. HRES-1/Rab41–121 increased the formation of LC3+ autophagosomes in resting cells, while other isoforms promoted autophagosome formation upon starvation. HRES-1/Rab4, HRES-1/Rab41–121, HRES-1/Rab4Q72L and HRES-1/Rab4S204Q promoted the accumulation of mitochondria during starvation. The specificity of HRES-1/Rab4-mediated mitochondrial accumulation is indicated by its abrogation by dominant-negative HRES-1/Rab4S27N mutation. The formation of interconnected mitochondrial tubular networks was markedly enhanced by HRES-1/Rab4Q72L upon starvation, which may contribute to the retention of mitochondria during autophagy. The present study thus indicates that HRES-1/Rab4 regulates autophagy through promoting the formation of LC3+ autophagosomes and the preservation of mitochondria.
Highlights
Autophagy is a complex stress-induced catabolic process that breaks down and recycles organelles and cytoplasmic materials [1]
The colocalization of HRES-1/Rab4 and light chain 3 (LC3) was investigated in HeLa cells transfected with LC3 fused to fluorescent protein 650 (FP650) (FP650-LC3) and HRES-1/Rab4 isoforms, including wild-type HRES-1/Rab4, HRES-1/Rab4S27N, HRES-1/Rab4Q72L, HRES-1/Rab4S204Q, and C-terminally truncated HRES-1/Rab41–121, each tagged to the C-terminus of enhanced green fluorescent protein (eGFP) (Fig. 1)
The present study provides evidence that the endosomal recycling regulator small GTPase HRES-1/Rab4 colocalizes with the autophagosomal membrane component LC3 and mitochondria, and, it promotes the colocalization between LC3 and mitochondria
Summary
Autophagy is a complex stress-induced catabolic process that breaks down and recycles organelles and cytoplasmic materials [1]. A new organelle, the autophagosome, is assembled from membrane components of mitochondria [3], plasma membrane [4] and endoplasmic reticulum [5]. Recycling endosomes have been suggested to contribute to the biogenesis of autophagosomes [6]. A signature component of the autophagosome membrane is microtubule-associated protein light chain 3 (LC3) [7], which has been widely identified as a binding partner of endosomal traffic regulator Rab GTPases [8,9]. The early endosomal marker Rab has been implicated in autophagosome formation [6], while the late endosomal Rab contributes to endosome maturation [9] and progression [10]
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