Revealing potential Rab proteins participate in regulation of secretory autophagy machinery

Abstract

Autophagy can be classified as degradative and secretory based on distinct functions. The small GTPase proteins Rab8a and Rab37 are responsible for secretory autophagy-mediated exocytosis of IL-1β, insulin, and TIMP1 (tissue inhibitor of 54 metalloproteinase 1). Other Rab family members participating in secretory autophagy are poorly understood. Herein, we identified 26 overlapped Rab proteins in purified autophagosomes of mouse pancreatic β-cell "Min-6" and human lung cancer cell "CL1-5-Q89L" with high secretory autophagy tendency by LC-MS/MS proteomics analysis. Six Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, Rab37, and Rab7a) were detected in autophagosomes of four cell lines, associating them with autophagy-related vesicle trafficking. We used CL1-5-Q89L cell line model to evaluate the levels of Rab proteins colocalization with autophagy LC3 proteins and presence in purified autophagosomes. We found five Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, and Rab37) are highly expressed in the autophagosome compared to the normal control by immunoblotting under active secretion conditions. However, only Rab8a, Rab35, and Rab37 showing high colocalization with LC3 protein by cofocal microscopy. Despite the discrepancy between the image and immunoblotting analysis, our data sustains the speculation that Rab8a, Rab11b, Rab27a, Rab35, and Rab37 are possibly associated with the secretory autophagy machinery. In contrast, Rab7a shows low colocalization with LC3 puncta and low level in the autophagosome, suggesting it regulates different vesicle trafficking machineries. Our findings open a new direction toward exploring the role of Rab proteins in secretory autophagy-related cargo exocytosis and identifying the cargoes and effectors regulated by specific Rab proteins.

Keywords: Rab protein; secretory autophagy; unconventional protein secretion.

FIGURE 1

Identification of 26 Rab proteins in the purified autophagosomes (APs) of mouse Min‐6 and human lung cancer CL1‐5‐Q89L under stimulation of secretory autophagy. The proteins in the purified APs of Min‐6 and CL1‐5‐Q89L cells were analyzed by LC–MS/MS proteomics analysis. We identified 26 Rab family proteins in 3012 overlapped proteins between Min‐6 and CL1‐5‐Q89L cells.

FIGURE 2

Colocalization of six autophagy‐related Rab and LC3 proteins in lung cancer CL1‐5 and CL1‐5‐Q89L cells under active secretory conditions. The cells were immunostaining with FITC‐conjugated anti‐LC3 antibody (green) or rhodamine‐conjugated anti‐Rab antibodies (red): (A) Rab8a, (B) Rab27a, (C) Rab37, (D) Rab11b, (E) Rab35, and (F) Rab7a at 24 h after starvation conditions. Scale bar = 5 μm. Green LC3 puncta represent autophagosomes, and red puncta represent Rab‐harbored vesicles. The colocalization of LC3 and Rab proteins was shown as a yellow color. For quantification, yellow immunofluorescence intensity was divided by red intensity for groups A, D, and E and green intensity for groups B, C, and F to calculate the percentage of LC3‐Rab colocalization. DAPI staining represents nuclei. ns: no significance, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

FIGURE 3

The levels of six autophagy‐related Rab proteins in the purified autophagosomes (APs) of lung cancer CL1‐5 (V) and CL1‐5‐Q89L (Q) cells under active secretory conditions. We compared six autophagy‐related Rab proteins in the post‐nucleus supernatant (PNS) and AP fractions of CL1‐5 (V) and CL1‐5‐Q89L (Q) cells under serum starvation conditions by immunoblotting using specific antibodies. LC3II is the marker of the AP, and calreticulin is the marker of ER. The numbers under the protein bands represent fold changes in protein levels compared to the control (AP‐V). An equal amount of protein (30 μg/well) from each fraction was loaded into the gel. V: CL1‐5 vector control: harboring the vector plasmid; Q: CL1‐5‐Q89L: harboring active‐form Rab37 plasmid.